Benzoate inductible promoters

ABSTRACT

Benzoate inducible promoters and promoter systems are disclosed, and uses thereof. Polynucleotides disclosing Benzoate Response Elements are also disclosed.

The present Application claims priority to U.S. Provisional ApplicationSer. No. 60/426,060 filed Nov. 13, 2002 and U.S. Provisional ApplicationSer. No. 60/425,760 filed Nov. 12, 2002, both of which are hereinincorporated by reference.

FIELD OF THE INVENTION

Benzoate inducible promoters and promoter systems are disclosed, anduses thereof. Polynucleotides disclosing Benzoate Response Elements arealso disclosed.

BACKGROUND OF THE INVENTION

The application of genetic engineering techniques to plants promises torevolutionize plant agriculture. One result of this revolution would bethe ability to control gene expression in plants. For example, pests anddiseases have been controlled by applying pesticides or biocides to cropplants; however, application of chemicals to plants affects more thanthe plant pests or diseases whose control is desired, and poses ageneral risk to the environment, often with deleterious consequences. Inan effort to ameliorate these risks, transgenic plants have beenrecently developed to constitutively express insect resistance genes ordisease resistance genes. However, constitutive expression means thatthese resistance genes are always expressed, not just when and where andto what level they are needed; such general expression can represent ametabolic drain on a plant, with consequent decreased productivity, orit can be too low to be effective. Moreover, constitutive expression ofa protein may not be desirable, particularly if this protein interfereswith the early stages of plant development. In other instances, highprotein levels cause toxicity to the plant.

Thus, in the last few years, there has been increasing interest amongplant scientists on means to precisely control the location, timing andlevel of expression of transgenes in plants, as well as of endogenousgenes. Controlling the location of gene expression, or a more precisecontrol of transgene expression in a specific plant tissue, has beenaccomplished by means of using one of several different tissue-specificpromoters. Controlling the timing and level of gene expression, ortemporal and quantitative control of expression, could be accomplishedby inducing gene expression upon the application of a specific stimulus.Such inducible gene expression would have numerous practicalapplications; for example, turning on engineered plant defense genesonly upon attack of a pathogen or insect predator could save millions ofdollars in pesticide application, as well as decrease unwanted adverseenvironmental effects. Inducing gene expression would also be a powerfulresearch tool, where it could be used in studies ranging from examiningphenotypes associated with specific gene expression to investigations ofgene interactions in plants.

One type of inducible promoter is a chemically inducible promoters.These are synthetic promoter systems often constructed by combiningknown regulator elements whose activity is modulated by the presence ofchemical effectors.

A chemically inducible promoter preferably satisfies several criteria tobe useful in an agricultural application. Such criteria includesufficient stability and relative non-toxicity of chemical inducers.Chemically-induced gene promoters have been isolated, but none of theseare suitable for practical application because of the nature of thechemical inducers. The chemicals are either too volatile (such asethanol) or toxic to the environment or the plants to which it isapplied (such as copper ions, antibiotics, or steroids).

Thus, what is needed are inducible promoters that are activated bychemicals which are sufficiently stable and non-toxic to theenvironment, including the plants to which they are applied. Preferablysuch chemical inducers can also be easily applied to large acreages ofcrop plants; even more preferably, such chemical inducers areinexpensive.

SUMMARY OF THE INVENTION

The present invention provides inducible promoters that are activated bychemicals that are stable and non-toxic to the environment, includingthe organisms, and in particular to plants, to which they are applied.The present invention also provides chemically inducible promoters wherethe chemical inducers can also be easily applied to large acreages ofcrop plants, and further where the chemical inducers are inexpensive.

The present invention provides novel chemically inducible promoters,promoter response elements, and promoter systems, modification of thepromoters for use in host cells, and use of the promoters and/ormodified promoters and promoter response systems in host cells tocontrol gene expression, where the inducing chemical is both stable andnon-toxic. An exemplary inducible promoter was isolated from Aspergillusniger. Specifically, a novel chemically inducible promoter and promotersystem identified in Aspergillus niger is induced by benzoate andrelated compounds.

Thus, the present invention provides novel benzoate inducible promoters,promoter response elements, modified or hybrid benzoate induciblepromoters, and promoter systems. In some embodiments, the presentinvention provides an isolated nucleic acid sequence of a benzoateinducible promoter region, where the promoter region is induced by thepresence of benzoate and/or related chemicals (e.g., benzoate mimetics).In particular embodiments, the present invention provides an isolatednucleic acid sequence of a benzoate inducible promoter region from afungus; in further particular embodiments, the benzoate induciblepromoter region is from Aspergillus niger. In other particularembodiments, the benzoate inducible promoter region is from a bphA gene;in further particular embodiments, the bphA gene is from Aspergillusniger.

In particular embodiments, the present invention provides an isolatedDNA molecule selected from the group consisting of the nucleic acidsequence comprising positions −1 to −531 of SEQ ID NO: 1 (SEQ ID NO:6),a 0.32 kb fragment (SEQ ID NO:2), comprising positions −199 to −521 ofSEQ ID NO:1), a 200 bp fragment (SEQ ID NO:3), comprising positions −331to −531 of SEQ ID NO:1, BREF51 (SEQ ID NO:4), comprising positions −357to −407 within SEQ ID NO:1, BRE6 (SEQ ID NO:5) comprising a 6 bpsequence TAGTCA, repeated twice in BREF51 but shown most active atposition −365 to −370, any fragment of SEQ ID NO:4 that is at leastabout 20, or 30 base pairs in length and that also comprises a correctlypositioned SEQ ID NO:5; any fragment of SEQ ID NO:6 that is at leastabout 20 base pairs in length and that also comprises at least onebenzoate response element (SEQ ID NO:5) (FIG. 1).

In some embodiments, the present invention provides compositionscomprising: a) a benzoate inducible promoter comprising; i) a benzoateresponse element comprising at least one copy of BRE6 sequence TAGTCA(or complement thereof); and ii) a heterologous gene promoter, and b) anucleic acid sequence of interest, wherein the nucleic acid sequence ofinterest is operably linked to the benzoate inducible promoter (e.g.linked such that the benzoate inducible promoter can drive expression ofthe nucleic acid sequence of interest when exposed to benzoate typecompound such as benzoic acid or similar compound).

In certain embodiments, the heterologous gene promoter comprises aminimal gene promoter (e.g. at least a minimal plant promoter). Inparticular embodiments, the benzoate response element comprises afragment of SEQ ID NO:6 or SEQ ID NO:1, wherein the fragment is at least15 or at least 20 or at least 30 base pairs in length. In certainembodiments, the nucleic acid sequence of interest comprises aheterologous gene sequence.

In particular embodiments, the benzoate response element comprises atleast two copies of the BRE6 sequence TAGTCA (e.g. two copies, threecopies or ten copies). In certain embodiments, the benzoate responseelement comprises a first BRE6 sequence (TAGTCA) and a second BRE6sequence (TAGTCA), and said first and second BRE6 sequence are separatedby 29 bases (e.g. any 29 bases, or the 29 bases between the two BRE6sshown in FIG. 8). In other embodiments, the benzoate response elementfurther comprises palidromic sequence GTTGTGTACATAAC (SEQ ID NO:7). Insome embodiments, the benzoate response element comprises a sequenceselected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, and SEQ ID NO:8, or the complement thereof.

In additional embodiments, the heterologous gene promoter is a plantpromoter (e.g. a plant minimal promoter, or a CaMV 35S promoter). Inother embodiments, the benzoate inducible promoter and the nucleic acidsequence of interest are located on a vector. In some embodiments, thevector further comprises a nucleic acid sequence encoding atranscription factor (e.g. a transcription needed in order for thebenzoate response element to function as a benzoate inducible promoter).In some embodiments, the compositions of the present invention comprisea transcription factor or a vector encoding a transcription factor. Incertain embodiments, the transcription factor is GAD1 (see FIG. 16),GAD11 (see FIG. 17), a GAD1 homolog (see FIG. 18), a GAD11 homolog (seeFIG. 19), or a functional fragment of any one of these. In certainembodiments, the benzoate inducible promoter is a benzoate induciblehybrid promoter.

In some embodiments, the present invention provides a transgenic plantcomprising: a) a benzoate inducible promoter comprising; i) a benzoateresponse element comprising at least one copy of BRE6 sequence TAGTCA;and ii) a heterologous gene promoter, and b) a nucleic acid sequence ofinterest, wherein the nucleic acid sequence of interest is operablylinked to the benzoate inducible promoter. In certain embodiments, theplant, when contacted with a benzoate type compound such as benzoic acidis configured to express said nucleic acid sequence of interest (e.g. adisease or pest resistance gene). In certain embodiments, the transgenicplants of the present invention are growing in a field and benzoic acidor similar compound is sprayed on the plants (e.g. to induce theexpression of the nucleic acid sequence of interest).

In certain embodiment, the present invention provides a transgenic seedcomprising: a) a benzoate inducible promoter comprising; i) a benzoateresponse element comprising at least one copy of BRE6 sequence TAGTCA;and ii) a heterologous gene promoter, and b) a nucleic acid sequence ofinterest, wherein the nucleic acid sequence of interest is operablylinked to the benzoate inducible promoter.

In some embodiments, the present invention provides methods oftransfecting a cell comprising; a) providing: i) a vector comprising; A)a benzoate inducible promoter comprising; I) a benzoate response elementcomprising at least one copy of BRE6 sequence TAGTCA; and II) aheterologous gene promoter, and B) a nucleic acid sequence of interest,wherein the nucleic acid sequence of interest is operably linked to thebenzoate inducible promoter; and ii) a target cell; and b) contactingthe vector with the vector under conditions such that the target cell istransfected and expresses the nucleic acid sequence of interest when thetarget cell is contacted with benzoate type compound such as benzoicacid or similar compound. In certain embodiments, the target cell ispart of a plant.

In certain embodiments, the present invention provides nucleic acidsequences selected from the group consisting of SEQ ID NOs:16, 18, 35and 37 (See FIGS. 16-19). In other embodiments, the present inventionprovides fragments or variants of SEQ ID NOs:16, 18, 35, and 37. In someembodiments, the present invention provides amino acid sequencesselected from SEQ ID NOs:15, 17, 34 and 36 (See FIGS. 16-19). Inadditional embodiments, the present invention provides fragments andvariants of SEQ ID NOs:15, 17, 34, and 36.

In other embodiments, the present invention provides an isolated DNAmolecule comprising a benzoate inducible promoter which hybridizes underhigh stringency to any of the isolated DNA molecules described above.

In other embodiments, the present invention provides an isolated DNAmolecule comprising a promoter, wherein the promoter is a benzoateinducible promoter and comprises any of the DNA molecules describedabove. In further embodiments, the DNA molecule further comprises aheterologous gene is operably linked to the benzoate promoter. Infurther embodiments, the DNA molecule further comprises a terminationsequence.

In other embodiments, the present invention provides an isolated DNAmolecule comprising a benzoate inducible hybrid promoter, which hybridpromoter comprises at least one benzoate response element within aheterologous gene promoter region. In some further embodiments, theheterologous gene promoter region is a minimal gene promoter. Inparticular embodiments, the at least one benzoate response elementcomprises SEQ ID NO:5.

In other embodiments, the present invention provides an expressionvector comprising an isolated DNA molecule comprising a promoter,wherein the promoter is a benzoate inducible promoter and comprises anyof the benzoate inducible promoters or benozate inducible hybridpromoters above. In some further embodiments, the expression furthercomprises a cloning site such that a nucleic acid sequence of interestcan be inserted into the vector and operably linked to the promoter orhybrid promoter. In other further embodiments, the vector furthercomprises a heterologous gene operably linked to the benzoate promoteror benzoate inducible hybrid promoter. In yet other embodiments, any ofthe vectors described above further comprises a termination sequence.

In another aspect, the present invention provides a transgenic cellcomprising a heterologous benzoate inducible promoter, where thepromoter region is induced by the presence of benzoate and/or relatedchemicals. In particular embodiments, the benzoate inducible promoter isfrom a fungus; in further particular embodiments, the benzoate induciblepromoter region is from Aspergillus niger. In other particularembodiments, the benzoate inducible promoter region is from a bphA gene;in further particular embodiments, the bphA gene is from Aspergillusniger. In yet other embodiments, the present invention provides atransgenic cell comprising any of the DNA molecules described above. Inother embodiments, the present invention provides a transgenic cellcomprising any of the benzoate inducible promoters or benzoate induciblehybrid promoters described above, which promoters comprise any of theDNA molecules described above. In some embodiments, the cell is aeukaryotic cell; in other embodiments, the cell is a prokaryotic cell.In yet other embodiments, the cell is a plant cell or an animal cell.

In another aspect, the present invention provides a transgenic plant orplant seed comprising a heterologous benzoate inducible promoter region,where the promoter region is induced by the presence of benzoate and/orrelated chemicals. In particular embodiments, the benzoate induciblepromoter is from a fungus; in further particular embodiments, thebenzoate inducible promoter region is from Aspergillus niger. In otherparticular embodiments, the benzoate inducible promoter region is from abphA gene; in further particular embodiments, the bphA gene is fromAspergillus niger. In yet other embodiments, the present inventionprovides a transgenic plant or plant seed comprising any of the DNAmolecules described above. In other embodiments, the present inventionprovides a transgenic plant or plant seed comprising any of the benzoateinducible promoters or benozate inducible hybrid promoters describedabove, which promoters comprise any of the DNA molecules describedabove.

The present invention also provides a purified molecule, wherein themolecule is a transacting factor necessary and sufficient to induce anyof the benzoate inducible promoters or benzoate inducible hybridpromoters as described above. In some embodiments, the factor is atranscription factor (e.g. GAD1, a GAD1 homolog, GAD11, or a GAD11homolog; see FIGS. 16-19).

The present invention also provides a composition comprising a benzoateinducible promoter system, wherein the benzoate inducible promotersystem comprises: a benzoate inducible promoter or benzoate induciblehybrid promoter as described above; and at least one additional nucleicacid sequence encoding a factor necessary and sufficient for inductionof the benzoate inducible promoter by benzoate and/or related chemicals.In some further embodiments, the inducible promoter or inducible hybridpromoter is linked to a cloning site such that a nucleic acid sequenceof interest can be inserted into the cloning site and operably linked tothe promoter or hybrid promoter. In other further embodiments, theinducible promoter or inducible hybrid promoter is operably linked to aheterologous gene. In other embodiments, the at least one additionalnucleic acid sequence encoding a factor is operably linked to apromoter. In further embodiments, the promoter operably linked to the atleast one additional nucleic acid sequence encoding a factor is aconstitutive promoter. In other embodiments, the factor is atranscription factor. In other embodiments, the benzoate induciblepromoter or benzoate inducible hybrid promoter and the at least oneadditional nucleic acid sequence encoding a factor of the benzoateinducible promoter system as described above are part of an expressionvector; in some embodiments, the promoter and the at least one codingsequence are part of separate expression vectors; in other embodiments,the promoter and the at least one coding sequence are part of the sameexpression vector.

In another aspect, the present invention provides a method of expressinga nucleic acid sequence of interest in a cell, comprising: providing atransgenic cell comprising a nucleic acid sequence of interest operablylinked to any of the benzoate inducible promoters or benzoate induciblehybrid promoters described above; and growing the cell under conditionssuch that the nucleic acid sequence of interest is expressed in thecell. In some embodiments, the nucleic acid sequence of interestoperably linked to any of the benzoate inducible promoters or benzoateinducible hybrid promoters is in an expression vector. In otherembodiments, the cell further comprises at least one additional nucleicacid sequence encoding a factor necessary and sufficient for inductionof the benzoate inducible promoter by benzoate and/or related chemicals;in some further embodiments, the at least one additional nucleic acidsequence is in an expression vector. In yet other embodiments, the cellis a plant cell.

In another aspect, the present invention provides a method of expressinga nucleic acid sequence of interest in a cell, comprising: providing atransgenic cell comprising a nucleic acid sequence of interest operablylinked to any of the benzoate inducible promoters or benzoate induciblehybrid promoters described above; and exposing the cell to benzoate or arelated chemical such that the nucleic acid sequence of interest isexpressed in the cell. In some embodiments, the nucleic acid sequence ofinterest operably linked to any of the benzoate inducible promoters orbenzoate inducible hybrid promoters is in an expression vector. In otherembodiments, the cell further comprises at least one additional nucleicacid sequence encoding a factor necessary and sufficient for inductionof the benzoate inducible promoter by benzoate and/or related chemicals;in some further embodiments, the at least one additional nucleic acidsequence is in an expression vector. In yet other embodiments, the cellis a plant cell.

In yet another aspect, the present invention provides a method ofinducing expression of a nucleic acid sequence of interest in a cell,comprising: providing a transgenic cell comprising a nucleic acidsequence of interest operably linked to any of the benzoate induciblepromoters or benzoate inducible hybrid promoters described above; andexposing the cell to benzoate or a related chemical such that thenucleic acid sequence of interest is expressed in the cell. In someembodiments, the nucleic acid sequence of interest operably linked toany of the benzoate inducible promoters or benzoate inducible hybridpromoters is in an expression vector. In other embodiments, the cellfurther comprises at least one additional nucleic acid sequence encodinga factor necessary and sufficient for induction of the benzoateinducible promoter by benzoate and/or related chemicals; in some furtherembodiments, the at least one additional nucleic acid sequence is in anexpression vector. In yet other embodiments, the cell is a plant cell.

In another aspect, the present invention provides a method ofcontrolling expression of flowering in a flowering plant, comprising:providing a transgenic plant comprising a gene necessary and sufficientto control flowering operably linked to any of the benzoate induciblepromoters or benzoate inducible hybrid promoters described above,wherein the gene regulatory region is a plant gene regulatory region;and exposing the plant to benzoate or a related chemical such that thegene necessary and sufficient to control flowering is expressed in theplant.

In yet another aspect, the present invention provides a method ofcontrolling expression of flowering in a flowering plant, comprising:providing a transgenic plant comprising a gene necessary and sufficientto inhibit flowering operably linked to any of the benzoate induciblepromoters or benzoate inducible hybrid promoters described above,wherein the gene regulatory region is a plant gene regulatory region;and exposing the plant to benzoate or a related chemical such that thegene necessary and sufficient to inhibit flowering is expressed in theplant.

In certain embodiments, the present invention provides kits comprising;a) one or more of the compositions described above; and b) a benzoatetype chemical or instructions on how to use the compositions of thepresent invention to transform plant cells (or other cell types) suchthat they express a sequence of interest when exposed to a benzoate typechemical.

DESCRIPTION OF THE FIGURES

FIG. 1 shows: A. DNA sequence of 1.8 kbp from the A. niger bphA genepromoter region (SEQ ID NO:1). Probable TATA Box sequence is underlined.Numbers to the left refer to nucleotide position relative to thetranscription start point. Italicized bases correspond to the conservedputative ORF identified by ORF Finder. BREF51 (SEQ ID NO:4) is shaded ingray. The TAGTCA sequence (SEQ ID NO:5) within BREF51 shown to beinvolved in factor binding in EMSA is double underlined. The proposedBenzoic Acid Response Element from van den Brink et al. (2000), which issituated within an open-reading frame of an adjacent gene that is notinduced by benzoate, is boxed. FIG. 1B shows positions −119 to −521 (SEQID NO:2) of the bphA gene promoter region. FIG. 1C shows positions −331to −531 (SEQ ID NO:3) of the bphA gene promoter region. FIG. 1D showspositions −357 to −407 (SEQ ID NO:4; BREF51) of the bphA gene promoterregion. FIG. 1E shows positions −365 to −370 (SEQ ID NO:5; BRE6) of thebphA gene promoter region. FIG. 1F shows positions −1 to −531 (SEQ IDNO:6) of the bphA gene promoter region.

FIG. 2 shows a genomic DNA blot, where genomic DNA from plant or fungalsources is probed with α-³²P-dCTP-labeled BPH probe, and shows anabsence of bphA homologs from plant species. Restriction of genomic DNAsfrom A. niger and A. nidulans with Xba I gives single bands of 12 kb and10 kb, respectively. Restriction of the DNAs with BstE II gives bands at1.1 kb and 12 kb, respectively. Lanes: Ag, Aspergillus niger; An,Aspergillus nidulans; At, Arabidopsis thaliana; Os, Oryza sativa; Nt,Nicotiana tabacum.

FIG. 3 shows a time course of bphA induction in the presence of 8 mMbenzoic acid. Total RNA was electrophoresed on a formaldehyde-agarosegel, blotted onto Hybond-N membrane, and probed with the 32P-labeled BPHprobe. Lanes, C, control, non-induced; numbers refer to minutes ofincubation in the presence of benzoic acid before total RNA extraction.A. Autoradiogram of membrane; B. Ethidium bromide-stained total RNA gelis a loading control. Arrowheads point to 2.1 kbp-size bandcorresponding to the bphA message. The RNA was probed withα-³²P-dCTP-labeled BPH probe. Lanes: Cont is a non-induced control takenat time 0, whereas 5, 10, 20, and 40 are times in minutes after additionof 8 mM benzoic acid. Shown as a loading control is an ethidiumbromide-stained total RNA gel (panel B).

FIG. 4 shows RNA blots of transcription of the bphA gene in response todifferent chemical inducers. The results show that transcription of thebphA gene is induced by 8 mM benzoic acid, as well as by sodium benzoateand methyl benzoate at the same concentration. Benzyl alcohol andhydrocinnamic acid are not effective inducers of the bphA gene at 8 mMconcentration. Top panel: RNA blot, probed with the ³²P-labeled BPHprobe (arrowhead points to 2.1 kbp-size band corresponding to the bphAmessage); Bottom panel: EtBr-stained gel, as a loading control. Lanes:C, control, uninduced; BA, benzoic acid; NaB, sodium benzoate; MeB,methyl benzoate; BAIc, benzyl alcohol; HyAc, hydrocinnamic acid.

FIG. 5 shows mini-promoter fragments from the upstream region of thebphA gene examined for protein binding. Seven mini-promoter fragments(FR1-FR7) were prepared by PCR, and labeled with ³²P-dATP and T4Polynucleotide kinase for Electrophoretic Mobility Shift Assays. Numbersrefer to position of base pair in relation to transcription start point(+1). FR1 to FR7 refer to mini-fragments 1 to 7, respectively. The * inthis figure indicates the localization of the proposed Benzoic AcidResponse Element by van den Brink et al. (2000).

FIG. 6 shows results of protein binding to the mini-promoter fragmentsshown in FIG. 5. Electrophoretic mobility shift assay with four 260 to330 bp fragments of the 1.8 kb putative promoter region was performed inthe presence (+) and absence (−) of protein. Total protein (cytoplasmicand nuclear) of Aspergillus cells were incubated with ³²P-labeledfragments for 1 h, and the mixtures then separated by polyacrylamide gelelectrophoresis. Positive binding of a cytosolic protein is indicatedwhen the electrophoretic mobility of a portion of the DNA fragment isslowed significantly. Fragment 6, is the only fragment to bind potentialtranscription factors. The arrowhead points to the mobility-shifted bandfrom FR6. It is noted that FR5 (not FR6) contains the Benzoic AcidResponse Element originally proposed by Van den Brink et al. (2000).

FIG. 7 shows the results of Electrophoretic Mobility Shift Assays (EMSA)which identified a 51-bp fragment containing a Benzoate Response Element(BRE). This fragment consistently binds a factor present in a totalprotein extract from benzoate-induced A. niger mycelia. The arrow pointsto the mobility-shifted band. Lanes: 1, ³²P-labeled BRE; 2, ³²P-labeledBRE+total protein extract from benzoate-induced A. niger 3, same as in2+excess of “cold” BRE; 4, same as in 2+“cold” DNA fragment frompromoter region of unrelated gene.

FIG. 8 shows, in panel A, the bphA promoter region that was tested forpromoter activity. This region comprises the 1847 nucleotides upstreamfrom the start sequence of the bphA gene (the bphA gene was published byvan Gorcom et al., Mol. Gen. Genet., 223:192-197, 2000; and is Genebankaccession No. X52521). Subsequently, it was discovered that much of thisputative promoter sequence actually comprises an open reading flame ofan unidentified protein. A response element was initially identified byvan den Brink et al. ((2000) Mol. Gen. Genet. 263: 601-609) based upon aconsensus sequence between A. niger and A nidulans. The identificationof the response element by van den Brink et al. (2000) Mol. Gen. Genet.263: 601-609) is determined to be erroneous, as it is now known thatthis initially identified response element is located in the openreading frame of the putative promoter sequence (from positions −532 to−1847); A. nidulans also contains this open-reading flame sequence inthe putative promoter sequence of the bphA gene. The entire 1847 bpsuspected promoter region, and fragments thereof generated by PCR, weretested for electrophoretic mobility shift activity (as described inFIGS. 5 and 6), and 0.4 kb Fragment 6 (FR6) was the only fragment of thesuspected promoter to exhibit shift activity. FR6 was fragmented furtherand only fragments containing a 51 bp sequence (as shown in panel B, SEQID NO:4) demonstrated EMSA shift activity (as described in FIG. 7). Thisfragment alone was one of the smallest fragment shown able to exhibit anelectrophoretic mobility shift, indicating the attachment of atranscription factor in vitro. Analysis of this sequence revealed a pairof 6 bp repeat sequences subsequently shown to be the suspected BenzoateResponse Element (BRE6); the sequence TAGTCA (SEQ ID NO:5). At least oneof these putative BREs is contemplated as necessary and sufficient foractivity (e.g. the one on the right in panel B). The 51 bp sequence (SEQID NO:4) is designated as BRE-containing fragment (BREF51).

FIG. 9 shows the benzoic acid-dependent GFP expression in A. nidulansGR5 strain carrying bphA promoter deletions. Panels a, b, c, g, h, andare bright field images of the fungal mycelia, and panels d, e, f, j, k,and l are corresponding images of GFP fluorescence; panels a, d, g, andj correspond to −1 to −1847 promoter sequence (SEQ ID NO:1)-GFP strain,panels b, e, h, and k correspond to −1 to −531 promoter sequence (SEQ IDNO:6)-GFP, panels c, f, i, and l correspond to −1 to −331 promotersequence-GFP; a to f, control, uninduced mycelia; g to l, myceliainduced with 8 mM benzoic acid for 5 h. This experiment shows that notonly transcriptional factor binding, but actual gene expression requiresthe BRE6 within BREF51, and included 200 bp from −332 to −531 (FIG. 9).

FIG. 10 shows an Electrophoretic Mobility Shift Assay (EMSA) of modifiedBREF51 sequences (SEQ ID NO:4) in which site-directed mutagenesis wasused to change either of the two TAGTCA sequences, delete the sequenceentirely or modify sequences flanking the downstream TAGTCA. The − and +symbols indicate labeled modified fragments incubated in the absence (−)or in the presence (+) of total protein extracts from A. niger cells.Control, indicates labeled BREF51 incubated in the absence (−) or in thepresence (+) of total protein extracts. The almost perfect palindromicsequence (SEQ ID NO:7) is boxed. The EMSA shows clearly that onlychanges to downstream TAGTCA (−364 to −369) abolish the vast majority ofthe binding.

FIG. 11 shows the results of an SDS-PAGE analysis of the fractionsobtained from an Affinity Chromatography experiment using BRE as theaffinity ligand. The gel was silver stained, and a protein ofapproximately 46 kD can be observed in the eluted fraction (arrow).Lanes: 1, supernatant fraction; 2, 1^(st) wash fraction; 3, 2^(nd) washfraction; 4, 3^(rd) wash fraction; 5, 4^(th) wash fraction; 6, elutedfraction; M, molecular weight marker (kDa).

FIG. 12 shows proposed working models for the mechanism of benzoateinduction of the bphA gene. It is noted that it is not necessary tounderstand what mechanism causes benzoate induction of the bphA gene topractice the present invention. In the model shown in panel A, benzoatefirst binds to a constitutive transcription factor (TF), and the complexbinds to the Benzoate Responsive Element (BRE) in the promoter region toactivate transcription. In the model shown in panel B, benzoate binds toa transcriptional repressor and induces dissociation. In the model shownin panel C, the benzoate binds to a membrane surface receptor thatinitiates the release of a specific TF that induces the bphA gene alongwith other genes required for benzoate uptake and metabolism. In themodel shown in panel D, the activation of the membrane surface receptorresults in release of a factor that induces dissociation of a repressorelement.

FIG. 13 shows an EMSA of BREF51 (SEQ ID NO:4) in the presence of totalprotein extracts from nine (of 17 total) selected yeast GAD clones.Lanes, Control−, labeled BREF51 only; Control+, labeled BREF51 plus 1 mgof total protein extracts from A. niger; GAD, labeled BREF51 plus 5 mgof total protein extracts from GAD clones (numbers refer to clones asdescribed on Table 1). Arrowhead points to control mobility shiftedband. Of the total of 17 clones, only two clones (GAD1 and GAD11), gavepositive mobility shifts, and they were in the size range observed withthe total protein extracts. The low binding was the result of very smallamounts of recombinant protein available. The function of the protein ofthe GAD1 gene or its homologs in the A. nidulans and Neurospora crassagenomes is not known. GAD1 (see FIG. 16) is rich in serine residues andpredicted by PSORTII to contain a nuclear localization signal. Thus, theprotein has the essential characteristics of a transcriptionalregulator. GAD11 (see FIG. 17) is weakly similar to a Drosophilamelanogaster homeotic gene regulator and to a Caenorhabditis elegansnuclear-targeted protein.

FIG. 14 shows the expression of GAD1 and GAD11 genes in response tobenzoic acid. Total RNA samples isolated from uninduced (−) and inducedfor 5 h in the presence of 8 mM benzoic acid (+) were reversetranscribed using Oligo (dT) primers, and cDNAs amplified using GAD1 andGAD11 gene-specific primers. M, 100 bp molecular weight marker; M′, 1kbp molecular weight marker. C, negative control of RT-PCR. Theexpression of GAD1 is constitutive, whereas the expression of GAD11 isslightly down-regulated. Expression of a benzoate-activation regulatorin the absence of the inducing molecule is expected, but thedown-regulation of GAD11 suggests that it functions as a repressor,although an understanding of this is not necessary to practice thepresent invention.

FIG. 15 shows an embodiment of the benzoate inducible hybrid promotersystem. The first DNA construct is the Inducible Promoter itself (panelA), with 3 or more copies of a BRE of the present invention (e.g. BREF51or other BRE containing a first and/or second copy of BRE6 (SEQ IDNO:5), fused to the TATA-box of the Cauliflower Mosaic Virus 35Spromoter (35S TATA). A polycloning site is engineered between the 35STATA promoter sequence and the Nopaline Synthase (nos) terminator forease of manipulation. This hybrid promoter drives expression of anydownstream gene of interest in a benzoate inducible manner. The nosterminator is placed downstream of the coding region and, in addition tostop codons in every reading frame, contains a polyadenylation signalsequence. The second DNA construct encodes the Transacting Factor orTranscriptional Factor (panel B), which is constitutively expressed byusing a full-length CaMV35S promoter. A chimeric transcription factorconstruct may also be employed that consists of a combination ofmodules. The first module is the Nuclear Localization Signal (NLS) fromthe 5V40 viral protein. The Activation Domain (AD) of the Herpes SimplexViral Protein 16 (VP16) constitutes the second module. The third andfinal module have the BREF51 DNA binding domain (BRE DBD) fused to thebenzoate receptor domain (Benz Rec), both identified from the A. nigertranscription factor. The nos terminator sequence is also placeddownstream of this construct.

FIG. 16 shows the amino acid sequence (SEQ ID NO:15) and nucleic acidsequence (SEQ ID NO:16) of GAD1.

FIG. 17 shows the amino acid sequence (SEQ ID NO:17) and nucleic acidsequence (SEQ ID NO:18) of GAD11.

FIG. 18 shows the amino acid sequence (SEQ ID NO:34) and nucleic acidsequence (SEQ ID NO:35) of a GAD1 homolog.

FIG. 19 shows the amino acid sequence (SEQ ID NO:36) and nucleic acidsequence (SEQ ID NO:37) of a GAD11 homolog.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases as used herein are defined below:

The term “plant” is used in it broadest sense. It includes, but is notlimited to, any species of woody, ornamental or decorative, crop orcereal, fruit or vegetable plant, and photosynthetic green algae (forexample, Chlamydomonas reinhardtii). It also refers to a plurality ofplant cells which are largely differentiated into a structure that ispresent at any stage of a plant's development. Such structures include,but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.The term “plant tissue” includes differentiated and undifferentiatedtissues of plants including those present in roots, shoots, leaves,pollen, seeds and tumors, as well as cells in culture (for example,single cells, protoplasts, embryos, callus, etc.). Plant tissue may bein planta, in organ culture, tissue culture, or cell culture. The term“plant part” as used herein refers to a plant structure or a planttissue. The term “seed” as used herein includes all tissues which resultfrom the development of a fertilized plant egg; thus, it includes amatured ovule containing an embryo and stored nutrients, as well as theintegument or integuments differentiated as the protective seed coat, ortesta. The nutrients in seed tissues may be stored in the endosperm orin the body of the embryo, notably in the cotyledons, or both.

The term “crop” or “crop plant” is used in its broadest sense. The termincludes, but is not limited to, any species of plant or algae edible byhumans or used as a feed for animals or used, or consumed by humans, orany plant or algae used in industry or commerce.

The term plant cell “compartments or organelles” is used in its broadestsense. The term includes but is not limited to, the endoplasmicreticulum, Golgi apparatus, trans Golgi network, plastids, sarcoplasmicreticulum, glyoxysomes, mitochondrial, chloroplast, and nuclearmembranes, and the like.

The term “benzoate para-hydroxylase (BPH)” refers to an enzyme whichcatalyzes the hydroxylation of the aromatic ring of benzoic acid at thepara position. The enzyme belongs to the class of cytochrome P450monooxygenases (CYP53A1). In the fungus Aspergillus niger, the enzymecatalyzes the first of a series of steps by which A. niger catabolizesbenzoic acid, and is thus able to utilize benzoic acid as a carbonsource.

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and are used interchangeably. A“protein” or “polypeptide” encoded by a gene is not limited to the aminoacid sequence encoded by the gene, but includes post-translationalmodifications of the protein.

Where the term “amino acid sequence” is recited herein to refer to anamino acid sequence of a protein molecule, “amino acid sequence” andlike terms, such as “polypeptide” or “protein” are not meant to limitthe amino acid sequence to the complete, native amino acid sequenceassociated with the recited protein molecule. Furthermore, an “aminoacid sequence” can be deduced from the nucleic acid sequence encodingthe protein.

The term “portion” or “fragment” when used in reference to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino sequence minus one amino acid.

The term “chimera” when used in reference to a polypeptide refers to theexpression product of two or more coding sequences obtained fromdifferent genes, that have been cloned together and that, aftertranslation, act as a single polypeptide sequence. Chimeric polypeptidesare also referred to as “hybrid” polypeptides. The coding sequencesincludes those obtained from the same or from different species oforganisms.

The term “fusion” when used in reference to a polypeptide refers to achimeric protein containing a protein of interest joined to an exogenousprotein fragment (the fusion partner). The fusion partner may servevarious functions, including enhancement of solubility of thepolypeptide of interest, as well as providing an “affinity tag” to allowpurification of the recombinant fusion polypeptide from a host cell orfrom a supernatant or from both. If desired, the fusion partner may beremoved from the protein of interest after or during purification.

The term “homolog” or “homologous” when used in reference to apolypeptide refers to a high degree of sequence identity between twopolypeptides, or to a high degree of similarity between thethree-dimensional structure or to a high degree of similarity betweenthe active site and the mechanism of action. In a preferred embodiment,a homolog has a greater than 60% sequence identity, and more preferablygreater than 75% sequence identity, and still more preferably greaterthan 90% sequence identity, with a reference sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (forexample, 99 percent sequence identity). Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.

The terms “variant” and “mutant” when used in reference to a polypeptiderefer to an amino acid sequence that differs by one or more amino acidsfrom another, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One may make variants of the BREs andtranscription factors described herein (with functionality of thesevariants tested as candidates in the protocols described in the Examplesbelow). One type of conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine. More rarely, a variant mayhave “non-conservative” changes (for example, replacement of a glycinewith a tryptophan). Similar minor variations may also include amino aciddeletions or insertions in other words, additions), or both. Guidance indetermining which and how many amino acid residues may be substituted,inserted or deleted without abolishing biological activity may be foundusing computer programs well known in the art, for example, DNAStarsoftware. Variants can be tested in functional assays. Preferredvariants have less than 10%, and preferably less than 5%, and still morepreferably less than 2% changes (whether substitutions, deletions, andso on).

The term “gene” refers to a nucleic acid (for example, DNA or RNA)sequence that comprises coding sequences necessary for the production ofan RNA, and/or ultimately a polypeptide or its precursor. A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (for example, enzymatic activity, ligand binding,signal transduction, etc.) of the polypeptide are retained. The term“portion” when used in reference to a gene refers to fragments of thatgene. The fragments may range in size from a few nucleotides to theentire gene sequence minus one nucleotide. Thus, “a nucleotidecomprising at least a portion of a gene” may comprise fragments of thegene or the entire gene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “heterologous” when used in reference to a gene refers to agene that is not in its natural environment (in other words, has beenaltered by the hand of man). For example, a heterologous gene includes agene from one species introduced into another species. A heterologousgene also includes a gene native to an organism that has been altered insome way (for example, mutated, added in multiple copies, linked to anon-native promoter or enhancer sequence, etc.). Heterologous genes maycomprise plant gene sequences that comprise cDNA forms of a plant gene;the cDNA sequences may be expressed in either a sense (to produce mRNA)or anti-sense orientation (to produce an anti-sense RNA transcript thatis complementary to the mRNA transcript). Heterologous genes aredistinguished from endogenous plant genes in that the heterologous genesequences are typically joined to nucleotide sequences comprisingregulatory elements such as promoters that are not found naturallyassociated with the gene for the protein encoded by the heterologousgene or with plant gene sequences in the chromosome, or are associatedwith portions of the chromosome not found in nature (for example, genesexpressed in loci where the gene is not normally expressed).

The term “oligonucleotide” refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. The term “polynucleotide” refers to a molecule comprised tomore than two deoxyribonucleotides or ribonucleotides, and is typicallylonger than an oligonucleotide. However, the terms “oligonucleotide,”“polynucleotide,” and “nucleic acid” are often used interchangeably. Thelength of an oligonucleotide is expressed as a number of base pairs (bp)or nucleotides. Although generally, nucleotides refer to the sensestrand, whereas base pair refers to the complementary base on theantisense strand that is understood for DNA, as used herein the terms“base pairs” or “nucleotides” to express the length of anoligonucleotide are used interchangeably. Also as the terms are usedherein, it is understood that a length expressed in base pairs does notmean that the molecule must be double stranded, but may exist as pairedor unpaired. Likewise, a length expressed as a number of nucleotidesdoes not mean that the molecule may not be double stranded, or in anyother form.

The term “nucleotide sequence of interest” or “nucleic acid sequence ofinterest” refers to any nucleotide sequence (for example, RNA or DNA),the manipulation of which may be deemed desirable for any reason (forexample, treat disease, confer improved qualities, etc.), by one ofordinary skill in the art. Such nucleotide sequences include, but arenot limited to, coding sequences of structural genes (for example,reporter genes, selection marker genes, oncogenes, drug resistancegenes, growth factors, etc.), and non-coding regulatory sequences whichdo not encode an mRNA or protein product (for example, promotersequence, polyadenylation sequence, termination sequence, enhancersequence, etc.).

The term “structural” when used in reference to a gene or to anucleotide or nucleic acid sequence refers to a gene or a nucleotide ornucleic acid sequence whose ultimate expression product is a protein(such as an enzyme or a structural protein), or an rRNA, an sRNA, atRNA, etc.

The term “fragment” or “portion” when used in reference to a anoligonucleotide sequence or nucleic acid sequence refers to a length ofthe sequence which is less than the entire length is it occurs naturally(for example, as a DNA, RNA, or cDNA molecule). The fragments may rangein size from a few nucleotides to the entire nucleic sequence minus onenucleotide. Thus, “a nucleotide comprising at least a portion of a gene”may comprise fragments of the gene or the entire gene

The term “an oligonucleotide having a nucleotide sequence encoding agene” or “a nucleic acid sequence encoding” a specified gene productrefers to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a geneproduct. The coding region may be present in either a cDNA, genomic DNAor RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (in other words, the sense strand) or double-stranded.Suitable control elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in expression vectors of thepresent invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

The terms “complementary” and “complementarity” refer to polynucleotides(in other words, a sequence of nucleotides) related by the base-pairingrules. For example, for the sequence “A-G-T,” is complementary to thesequence “T-C-A.” Complementarity may be “partial,” in which only someof the nucleic acids' bases are matched according to the base pairingrules. Or, there may be “complete” or “total” complementarity betweenthe nucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology or completehomology (in other words, identity). “Sequence identity” refers to ameasure of relatedness between two or more nucleic acids or proteins,and is given as a percentage with reference to the total comparisonlength. The identity calculation takes into account those nucleotide oramino acid residues that are identical and in the same relativepositions in their respective larger sequences. Calculations of identitymay be performed by algorithms contained within computer programs suchas “GAP” (Genetics Computer Group, Madison, Wis.) and “ALIGN” (DNAStar,Madison, Wis.). A partially complementary sequence is one that at leastpartially inhibits (or competes with) a completely complementarysequence from hybridizing to a target nucleic acid is referred to usingthe functional term “substantially homologous.” The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (in other words, the hybridization)of a sequence which is completely homologous to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (in other words, selective) interaction. Theabsence of non-specific binding may be tested by the use of a secondtarget which lacks even a partial degree of complementarity (forexample, less than about 30% identity); in the absence of non-specificbinding the probe will not hybridize to the second non-complementarytarget.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (in other words, a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (in other words, gaps) of 20percent or less as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by the local homology algorithm of Smith andWaterman (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)) by thehomology alignment algorithm of Needleman and Wunsch (Needleman andWunsch, J. Mol. Biol. 48:443 (1970)), by the search for similaritymethod of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci.(U.S.A.) 85:2444 (1988)), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection, and the best alignment (in otherwords, resulting in the highest percentage of homology over thecomparison window) generated by the various methods is selected. Theterm “sequence identity” means that two polynucleotide sequences areidentical (in other words, on a nucleotide-by-nucleotide basis) over thewindow of comparison. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (for example, A, T, C, G, U, or I) occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison (in other words, the window size), and multiplyingthe result by 100 to yield the percentage of sequence identity. Theterms “substantial identity” as used herein denotes a characteristic ofa polynucleotide sequence, wherein the polynucleotide comprises asequence that has at least 85 percent sequence identity, preferably atleast 90 to 95 percent sequence identity, more usually at least 99percent sequence identity as compared to a reference sequence over acomparison window of at least 20 nucleotide positions, frequently over awindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison. The reference sequence may be a subset of a larger sequence,for example, as a segment of the full-length sequences of thecompositions claimed in the present invention.

The term “substantially homologous” when used in reference to adouble-stranded nucleic acid sequence such as a cDNA or genomic clonerefers to any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described below.

The term “substantially homologous” when used in reference to asingle-stranded nucleic acid sequence refers to any probe that canhybridize (in other words, it is the complement of) the single-strandednucleic acid sequence under conditions of low to high stringency asdescribed below.

The term “hybridization” refers to the pairing of complementary nucleicacids. Hybridization and the strength of hybridization (in other words,the strength of the association between the nucleic acids) is impactedby such factors as the degree of complementary between the nucleicacids, stringency of the conditions involved, the T_(m) of the formedhybrid, and the G:C ratio within the nucleic acids. A single moleculethat contains pairing of complementary nucleic acids within itsstructure is said to be “self-hybridized.”

The term “T_(m)” refers to the “melting temperature” of a nucleic acid.The melting temperature is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half dissociated intosingle strands. The equation for calculating the T_(m) of nucleic acidsis well known in the art. As indicated by standard references, a simpleestimate of the T_(m) value may be calculated by the equation:T_(m)=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1M NaCl (See for example, Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985)). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

The term “stringency” refers to the conditions of temperature, ionicstrength, and the presence of other compounds such as organic solvents,under which nucleic acid hybridizations are conducted. With “highstringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “low” stringency areoften required with nucleic acids that are derived from organisms thatare genetically diverse, as the frequency of complementary sequences isusually less.

“Low stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5×Denhardt's reagent (50×Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising5× SSPE, 0.1% SDS at 42EC when a probe of about 500 nucleotides inlength is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0× SSPE, 1.0% SDS at 42EC when aprobe of about 500 nucleotides in length is employed.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42EC in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1× SSPE, 1.0% SDS at 42EC when aprobe of about 500 nucleotides in length is employed.

It is well known that numerous equivalent conditions may be employed tocomprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (forexample, the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions that promote hybridization underconditions of high stringency (for example, increasing the temperatureof the hybridization and/or wash steps, the use of formamide in thehybridization solution, etc.).

The term “gene expression” refers to the process of converting geneticinformation encoded in a gene into RNA (for example, mRNA, rRNA, tRNA,or snRNA) through “transcription” of the gene (in other words, via theenzymatic action of an RNA polymerase), and into protein, through“translation” of mRNA. Gene expression can be regulated at many stagesin the process. “Up-regulation” or “activation” refers to regulationthat increases the production of gene expression products (in otherwords, RNA or protein), while “down-regulation” or “repression” refersto regulation that decrease production. Molecules (for example,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

The terms “in operable combination”, “in operable order” and “operablylinked” refer to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the transcription of agiven gene and/or the synthesis of a desired protein molecule isproduced. The term also refers to the linkage of amino acid sequences insuch a manner so that a functional protein is produced.

The term “regulatory element” refers generally to a genetic elementwhich controls some aspect of the expression of nucleic acid sequences.For example, under this definition, a promoter is a regulatory elementwhich facilitates the initiation of transcription of an operably linkedcoding region. More specifically, a regulatory element refers to activeoligonucleotide sequences within promoters, within introns, and withinthe 3′ untranslated sequences. Thus, under this more specificdefinition, a promoter may consist of a collection of several kinds ofelements. Other regulatory elements are splicing signals,polyadenylation signals, termination signals, etc.

The term “regulatory region” refers to a gene's 5′ transcribed butuntranslated regions, located immediately downstream from the promoterand ending just prior to the translational start of the gene.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” and “repressor” elements (discussed further below). Promotersand enhancers consist of short arrays of DNA sequences that interactspecifically with cellular proteins involved in transcription (Maniatis,et al., Science 236:1237, 1987). Promoter and enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inyeast, insect, mammalian and plant cells. Promoter and enhancer elementshave also been isolated from viruses and analogous control elements,such as promoters, are also found in prokaryotes. The selection of aparticular promoter and enhancer depends on the cell type used toexpress the protein of interest. Some eukaryotic promoters and enhancershave a broad host range while others are functional in a limited subsetof cell types (for review, see Voss, et al, Trends Biochem. Sci.,11:287, 1986; and Maniatis, et al., supra 1987).

The term “promoter region” refers to the region immediately upstream ofthe coding region of a DNA polymer, and is typically between about 500bp and 4 kb in length, and is preferably about 1 to about 1.5 kb inlength. A promoter region controls or regulates transcription of a geneto which it is operably linked, either naturally or by recombinantnucleic acid technology. A promoter region may include smaller sequenceswhich are effective to control or regulate transcription. One skilled inthe art can determine such smaller sequences by creating fragments ofdecreasing size from a promoter region, and operably linking suchfragments to a reporter gene, and determining expression of suchconstructs in transgenic tissue, as described further herein.

The terms “promoter element,” “promoter,” or “promoter sequence” referto a DNA sequence that is located at the 5′ end (in other wordsprecedes) of the coding region of a DNA polymer. The location of mostpromoters known in nature precedes the transcribed region. The promoterfunctions as a switch, activating the expression of a gene. If the geneis activated, it is said to be transcribed, or participating intranscription. Transcription involves the synthesis of mRNA from thegene. The promoter, therefore, serves as a transcriptional regulatoryelement and also provides a site for initiation of transcription of thegene into mRNA.

Promoters generally may be tissue specific or cell specific; morespecifically, promoters may contain elements that impart tissue or cellspecificity. The term “tissue specific” as it applies to a promoterrefers to a promoter that is capable of directing selective expressionof a nucleotide sequence of interest to a specific type of tissue (forexample, seeds) in the relative absence of expression of the samenucleotide sequence of interest in a different type of tissue (forexample, leaves). Tissue specificity of a promoter may be evaluated by,for example, operably linking a reporter gene to the promoter sequenceto generate a reporter construct, introducing the reporter constructinto the genome of a plant such that the reporter construct isintegrated into every tissue of the resulting transgenic plant, anddetecting the expression of the reporter gene (for example, detectingmRNA, protein, or the activity of a protein encoded by the reportergene) in different tissues of the transgenic plant. The detection of agreater level of expression of the reporter gene in one or more tissuesrelative to the level of expression of the reporter gene in othertissues shows that the promoter is specific for the tissues in whichgreater levels of expression are detected. The term “cell type specific”as applied to a promoter refers to a promoter which is capable ofdirecting selective expression of a nucleotide sequence of interest in aspecific type of cell in the relative absence of expression of the samenucleotide sequence of interest in a different type of cell within thesame tissue. The term “cell type specific” when applied to a promoteralso means a promoter capable of promoting selective expression of anucleotide sequence of interest in a region within a single tissue. Celltype specificity of a promoter may be assessed using methods well knownin the art, for example, immunohistochemical staining. Briefly, tissuesections are embedded in paraffin, and paraffin sections are reactedwith a primary antibody which is specific for the polypeptide productencoded by the nucleotide sequence of interest whose expression iscontrolled by the promoter. A labeled (for example, peroxidaseconjugated) secondary antibody which is specific for the primaryantibody is allowed to bind to the sectioned tissue and specific bindingdetected (for example, with avidin/biotin) by microscopy.

A promoter is “effective” as a tissue specific or cell type promoterwhen expression in the presence of the promoter is greater in the tissueor cell type than expression in the presence of the promoter in othertissues or cell types. Preferably, the greater level of expression is atleast about two-fold greater; more preferably, it is at least aboutfour-fold greater; and most preferably, it is at least about ten-foldgreater. An effective promoter may comprise all of the promoter region,or a modification or fragment of a promoter region, or a motif of apromoter region.

Promoters may be constitutive or inducible. The term “constitutive” whenmade in reference to a promoter means that the promoter is capable ofdirecting transcription of an operably linked nucleic acid sequence inthe absence of a stimulus (for example, heat shock, chemicals, light,etc.). Typically, constitutive promoters are capable of directingexpression of a transgene in substantially any cell and any tissue.Exemplary constitutive plant promoters include, but are not limited toSD Cauliflower Mosaic Virus (CaMV SD; see for example, U.S. Pat. No.5,352,605, incorporated herein by reference), mannopine synthase,octopine synthase (ocs), superpromoter (see for example, WO 95/14098),and ubi3 (see for example, Garbarino and Belknap, Plant Mol. Biol.24:119-127 (1994)) promoters. Such promoters have been used successfullyto direct the expression of heterologous nucleic acid sequences intransformed plant tissue.

In contrast, an “inducible” promoter is one which is capable ofdirecting a level of transcription of an operably linked nucleic acidsequence in the presence of a stimulus (for example, heat shock,chemicals, light, etc.) which is different from the level oftranscription of the operably linked nucleic acid sequence in theabsence of the stimulus.

The term “benzoate inducible promoter” refers to a promoter which isinduced by the presence of benzoate and/or related chemicals. Benzoateand related chemicals include but are not limited to benzoic acid andits salts (Na⁺, K⁺, NH₄ ⁺, etc.), and esterified forms, where theesterified are used to facilitate penetration of the esterified formsinto cells and once inside the cells are subsequently be de-esterifiedto yield active compound by the cell's own esterases. Thus, relatedchemicals include but are not limited to sodium benzoate and methylbenzoate. It is contemplated, in some embodiments, that featuresnecessary and sufficient for activity are a benzyl ring with onecarboxyl group. Derivatives with substitutions at the ortho- andmeta-positions of the ring reduce or abolish activity, whereassubstitutions at the para-position, such as para-aminobenzoate, aretolerated. It is noted that additional benzoate type compounds can belocated by screening a test benzoate type compound in an assay (e.g. asdescribed in the Examples below) known to be induced by benzoic acid. Ifthe test benzoate compound also induces the expression of a sequence(such as GFP) then the compound is a benzoate type compound and isuseful with the benzoate inducible promoters of the present invention.

The term “response element” refers to specific short DNA sequence(s) ina promoter of a eukaryotic gene which controls transcription of thatparticular gene

The term “benzoate response element (BRE)” refers to at least one shortoligonucleotide sequence that binds to at least one transacting factorwithin a benzoate inducible promoter in the presence of benzoate; it iscontemplated that the oligonucleotide sequence is necessary forattachment of a benzoate-inducing transcriptional factor within thepromoter. It is further contemplated that in some embodiments, thissequence comprises either one or a pair of 6 bp repeats of the sequenceTAGTCA. In some further embodiments, this pair of short repeatedsequences is found in BREF51, where BREF51 is a fragment from thebenzoate inducible promoter region of the bphA gene located from −1 to−350 (or −1 to −531) bp upstream of the transcriptional start point, andwhere BREF51 consistently binds a protein factor present in totalprotein extracts from benzoate-induced A. niger mycelia. BREF51 alsopossesses benzoate-inducing activity in vivo. In some embodiments,additional sequences surrounding the 6 bp sequence TAGTCA (SEQ ID NO:5)are included as part of the BRE (e.g. 1, 5, 10, 25 or 35 additionalbases upstream, downstream, or both upstream and downstream of SEQ IDNO:5 are included. In certain preferred embodiments, a fragment of SEQID NO:6 at least 20 base pairs in length that includes TAGTCA (SEQ IDNO:5) is employed. Fragments of SEQ ID NO:6 that would function as BREs(and that are at least 20 base pairs and contain TAGTCA) may be testedin the assays such as those shown in the Examples section below todetermine if they will function as effective BREs.

The term “transacting factor” refers to a regulatory protein which bindsto a specific short DNA sequence(s) in the regulatory region of aeukaryotic gene controlling transcription of that particular gene. It iscontemplated that in a benzoate inducible promoter, a transacting factoris a transcription factor. Transcription factors interact with RNApolymerase, and may interact with each other, to modulate transcription.In some embodiments, GAD1 or GAD11 (or both) serve as transcriptionfactors that interact with the BRE. Transcription factors may interactwith promoter elements as either “inducers” or “repressors”. Inducerstypically bind to a promoter only in the presence of the inducingsubstances, whereas repressors are bound to the promoter in the absenceof the inducer and binding of the inducing molecule releases therepressor to allow transcription (see, e.g, FIG. 12).

The term “benzoate inducible hybrid promoter (BIHP)” refers to a hybridpromoter comprising benzoate responsive elements within a generegulatory region, such that the hybrid promoter is functional in cellsand is responsive to benzoate and related chemical inducers, in thepresence of any necessary and sufficient transacting factors.

The term “benzoate inducible promoter system (BIPS)” refers to acombination of nucleotide sequences comprising a benzoate induciblepromoter of the present invention, including a benzoate inducible hybridpromoter, and at least one additional gene encoding a factor asnecessary, and sufficient for the induction of the promoter component bybenzoate and/or related chemicals. In some embodiments, the factor is atransacting factor; in further embodiments, a transacting factor is atranscription factor. The at least one additional gene encoding a factoris under control of a promoter, often a constitutive promoter.Preferably, the benzoate promoter system is present in at least oneexpression vector, where the expression vector(s) can be used totransfect a host cell, either transiently or stably.

The term “functional equivalence” and its grammatical variants when usedin reference to nucleic acid sequences means that the nucleic acidsequences are capable of induction by benzoate and/or related chemicalseither by themselves, or when incorporated into a hybrid promoter, whenoperably linked to a gene of interest.

The enhancer and/or promoter may be “endogenous” or “exogenous” or“heterologous.” An “endogenous” enhancer or promoter is one that isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer or promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (in otherwords, molecular biological techniques) such that transcription of thegene is directed by the linked enhancer or promoter. For example, anendogenous promoter in operable combination with a first gene can beisolated, removed, and placed in operable combination with a secondgene, thereby making it a “heterologous promoter” in operablecombination with the second gene. A variety of such combinations arecontemplated (for example, the first and second genes can be from thesame species, or from different species).

The term “naturally linked” or “naturally located” when used inreference to the relative positions of nucleic acid sequences means thatthe nucleic acid sequences exist in nature in the relative positions.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp.16.7-16.8). A commonly used splice donor and acceptor site is the splicejunction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsgenerally requires expression of signals directing the efficienttermination and polyadenylation of the resulting transcript.Transcription termination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly(A) site” or “poly(A) sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable, as transcripts lacking a poly(A) tail areunstable and are rapidly degraded. The poly(A) signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly(A) signal is one that is found naturally at the 3′ end of thecoding region of a given gene in the genome. A heterologous poly(A)signal is one which has been isolated from one gene and positioned 3′ toanother gene. A commonly used heterologous poly(A) signal is the SV40poly(A) signal. The SV40 poly(A) signal is contained on a 237 bpBamHI/BclI restriction fragment and directs both termination andpolyadenylation (Sambrook, supra, at 16.6-16.7).

The term “termination signal” or “termination sequence” refers to a 3′non-translated DNA sequence which functions in plant cells to cause theaddition of polyadenylated ribonucleotides to the 3′ end of an mRNAsequence transcribed from a gene; the gene may be an endogenous ornative gene, or it may be a heterologous gene. The termination sequencemay be endogenous or heterologous to the gene.

The term “vector” refers to nucleic acid molecules that transfer DNAsegment(s) from one cell to another. The term “vehicle” is sometimesused interchangeably with “vector.”

The terms “expression vector” or “expression cassette” refer to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences generally necessary for expression in prokaryotes usuallyinclude a promoter, an operator (optional), and a ribosome binding site,often along with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

The term “transfection” refers to the introduction of foreign DNA intocells. Transfection may be accomplished by a variety of means known tothe art including, but not limited to, calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, glass beads, electroporation, microinjection, liposomefusion, lipofection, protoplast fusion, viral infection, biolistics (inother words, particle bombardment) and the like. The term“transformation” and its grammatical variants (including but not limitedto transform, transformed, and transforming) is used interchangeablywith the term “transfection” and its grammatical variants (includingtransfect, transfected, and transfecting).

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 (1973)),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

The terms “infecting” and “infection” when used with a bacterium referto co-incubation of a target biological sample, (for example, cell,tissue, etc.) with the bacterium under conditions such that nucleic acidsequences contained within the bacterium are introduced into one or morecells of the target biological sample.

The term “Agrobacterium” refers to a soil-borne, Gram-negative,rod-shaped phytopathogenic bacterium which causes crown gall. The term“Agrobacterium” includes, but is not limited to, the strainsAgrobacterium tumefaciens, (which typically causes crown gall ininfected plants), and Agrobacterium rhizogens (which causes hairy rootdisease in infected host plants). Infection of a plant cell withAgrobacterium generally results in the production of opines (forexample, nopaline, agropine, octopine etc.) by the infected cell. Thus,Agrobacterium strains which cause production of nopaline (for example,strain LBA4301, C58, A208, GV3101) are referred to as “nopaline-type”Agrobacteria; Agrobacterium strains which cause production of octopine(for example, strain LBA4404, Ach5, B6) are referred to as“octopine-type” Agrobacteria; and Agrobacterium strains which causeproduction of agropine (for example, strain EHA105, EHA101, A281) arereferred to as “agropine-type” Agrobacteria.

The terms “bombarding, “bombardment,” and “biolistic bombardment” referto the process of accelerating particles towards a target biologicalsample (for example, cell, tissue, etc.) to effect wounding of the cellmembrane of a cell in the target biological sample and/or entry of theparticles into the target biological sample. Methods for biolisticbombardment are known in the art (for example, U.S. Pat. No. 5,584,807,the contents of which are incorporated herein by reference), and arecommercially available (for example, the helium gas-drivenmicroprojectile accelerator (PDS-1000/He, BioRad).

The term “microwounding” when made in reference to plant tissue refersto the introduction of microscopic wounds in that tissue. Microwoundingmay be achieved by, for example, particle bombardment as describedherein.

The term “transgene” refers to a foreign gene that is placed into anorganism by the process of transfection. The term “foreign gene” refersto any nucleic acid (for example, gene sequence) that is introduced intothe genome of an organism by experimental manipulations and may includegene sequences found in that organism so long as the introduced genedoes not reside in the same location as does the naturally-occurringgene.

The term “transgenic” when used in reference to a cell or organism (inother words, a “transgenic cell” or “transgenic organism”) refers to acell or organism that contains at least one heterologous or foreign genein it or in one or more of the cells of the organism. The term“transgenic” when used in reference to a plant or fruit or seed (inother words, a “transgenic plant” or “transgenic fruit” or a “transgenicseed”) refers to a plant or fruit or seed that contains at least oneheterologous or foreign gene in one or more of its cells. The term“transgenic plant material” refers broadly to a plant, a plantstructure, a plant tissue, a plant seed or a plant cell that contains atleast one heterologous gene in one or more of its cells.

The term “host cell” refers to any cell capable of replicating and/ortranscribing and/or translating a heterologous gene. Thus, a “host cell”refers to any eukaryotic or prokaryotic cell (for example, bacterialcells such as E. coli, yeast cells, mammalian cells, avian cells,amphibian cells, plant cells, fish cells, and insect cells), whetherlocated in vitro or in vivo. For example, host cells may be located in atransgenic animal.

The terms “transformants” or “transformed cells” include the primarytransformed cell and cultures derived from that cell without regard tothe number of transfers. All progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same functionality as screened for in the originallytransformed cell are included in the definition of transformants.

The term “selectable marker” refers to a gene which encodes an enzymehaving an activity that confers resistance to an antibiotic or drug uponthe cell in which the selectable marker is expressed, or which confersexpression of a trait which can be detected (for example., luminescenceor fluorescence). Selectable markers may be “positive” or “negative.”Examples of positive selectable markers include the neomycinphosphotrasferase (NPTII) gene which confers resistance to G418 and tokanamycin, and the bacterial hygromycin phosphotransferase gene (hyg),which confers resistance to the antibiotic hygromycin. Negativeselectable markers encode an enzymatic activity whose expression iscytotoxic to the cell when grown in an appropriate selective medium. Forexample, the HSV-tk gene is commonly used as a negative selectablemarker. Expression of the HSV-tk gene in cells grown in the presence ofgancyclovir or acyclovir is cytotoxic; thus, growth of cells inselective medium containing gancyclovir or acyclovir selects againstcells capable of expressing a functional HSV TK enzyme.

The term “reporter gene” refers to a gene encoding a protein that may beassayed. Examples of reporter genes include, but are not limited to,β-glucuronidase (GUS), luciferase (See for example, deWet et al., Mol.Cell. Biol. 7:725 (1987) and U.S. Pat. Nos., 6,074,859; 5,976,796;5,674,713; and 5,618,682; all of which are incorporated herein byreference), green fluorescent protein (for example, GenBank AccessionNumber U43284; a number of GFP variants are commercially available fromCLONTECH Laboratories, Palo Alto, Calif.), chloramphenicolacetyltransferase, β-galactosidase, alkaline phosphatase, and horseradish peroxidase.

The term “wild-type” when made in reference to a nucleic acid sequencerefers to a nucleic acid sequence which has the characteristics of thesequence isolated from a naturally occurring source. The term“wild-type” when made in reference to a gene product refers to a geneproduct which has the characteristics of a gene product isolated from anaturally occurring source. The term “naturally-occurring” as applied toan object refers to the fact that an object can be found in nature. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses) that can be isolated from a source innature and which has not been intentionally modified by man in thelaboratory is naturally-occurring. A wild-type gene is that which ismost frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. In contrast,the term “modified” or “mutant” when made in reference to a nucleic acidsequence (such as a regulatory sequence or a sequence encoding a gene)or to a gene product refers, respectively, to a nucleic acid sequence orto a gene product which displays modifications in sequence and/orfunctional properties (in other words, altered characteristics) whencompared to the wild-type gene or gene product. Modifications includeadditions or deletions of the units making up the nucleic acid sequenceor gene product (a unit is, for example, a nucleotide), or substitutionsof at least one of the units. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type nucleic acidsequence or gene product.

The term “antisense” refers to a deoxyribonucleotide sequence whosesequence of deoxyribonucleotide residues is in reverse 5′ to 3′orientation in relation to the sequence of deoxyribonucleotide residuesin a sense strand of a DNA duplex. A “sense strand” of a DNA duplexrefers to a strand in a DNA duplex which is transcribed by a cell in itsnatural state into a “sense mRNA.” Thus an “antisense” sequence is asequence having the same sequence as the non-coding strand in a DNAduplex. The term “antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene by interfering with theprocessing, transport and/or translation of its primary transcript ormRNA. The complementarity of an antisense RNA may be with any part ofthe specific gene transcript, in other words, at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence. Inaddition, as used herein, antisense RNA may contain regions of ribozymesequences that increase the efficacy of antisense RNA to block geneexpression. “Ribozyme” refers to a catalytic RNA and includessequence-specific endoribonucleases. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of preventing theexpression of the target gene, in that the protein the target genenormally encodes is not produced or is produced at a lower level than inthe absence of the antisense RNA transcripts.

The term “overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. The term “cosuppression” refers to theexpression of a foreign gene which has substantial homology to anendogenous gene resulting in the suppression of expression of both theforeign and the endogenous gene. As used herein, the term “alteredlevels” refers to the production of gene product(s) in transgenicorganisms in amounts or proportions that differ from that of normal ornon-transformed organisms.

The terms “Southern blot analysis” and “Southern gel blot analysis” and“Southern blot” and “Southern” refer to the analysis of DNA on agaroseor acrylamide gels in which DNA is separated or fragmented according tosize followed by transfer of the DNA from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized DNA is thenexposed to a labeled probe to detect DNA species complementary to theprobe used. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Press, NY), pp 9.31-9.58).

The term “Northern blot analysis” and “Northern gel blot analysis” and“Northern blot” and “Northern” refer to the analysis of RNA byelectrophoresis of RNA on agarose gels to fractionate the RNA accordingto size followed by transfer of the RNA from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized RNA is thenprobed with a labeled probe to detect RNA species complementary to theprobe used. Northern blots are a standard tool of molecular biologists(J. Sambrook, et al. (1989) supra, pp 7.39-7.52).

The terms “Western blot analysis” and “Western gel blot analysis” and“Western blot” and “Western” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. A mixture comprising at least one protein is first separatedon an acrylamide gel, and the separated proteins are then transferredfrom the gel to a solid support, such as nitrocellulose or a nylonmembrane. The immobilized proteins are exposed to at least one antibodywith reactivity against at least one antigen of interest. The boundantibodies may be detected by various methods, including the use ofradiolabeled antibodies.

The term “antigenic determinant” refers to that portion of an antigenthat makes contact with a particular antibody (in other words, anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (in other words, the “immunogen” used toelicit the immune response) for binding to an antibody.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature. Forexample, a given DNA sequence (for example, a gene) is found on the hostcell chromosome in proximity to neighboring genes; RNA sequences, suchas a specific mRNA sequence encoding a specific protein, are found inthe cell as a mixture with numerous other mRNA s which encode amultitude of proteins. However, isolated nucleic acid encoding aparticular protein includes, by way of example, such nucleic acid incells ordinarily expressing the protein, where the nucleic acid is in achromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid or oligonucleotide may be presentin single-stranded or double-stranded form. When an isolated nucleicacid or oligonucleotide is to be utilized to express a protein, theoligonucleotide will contain at a minimum the sense or coding strand (inother words, the oligonucleotide may single-stranded), but may containboth the sense and anti-sense strands (in other words, theoligonucleotide may be double-stranded).

The term “purified” refers to molecules, either nucleic or amino acidsequences, that are removed from their natural environment, isolated orseparated. An “isolated nucleic acid sequence” is therefore a purifiednucleic acid sequence. “Substantially purified” molecules are at least60% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated. Asused herein, the term “purified” or “to purify” also refer to theremoval of contaminants from a sample. The removal of contaminatingproteins results in an increase in the percent of polypeptide ofinterest in the sample. In another example, recombinant polypeptides areexpressed in plant, bacterial, yeast, or mammalian host cells and thepolypeptides are purified by the removal of host cell proteins; thepercent of recombinant polypeptides is thereby increased in the sample.

The term “sample” is used in its broadest sense. In one sense it canrefer to a plant cell or tissue. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

DESCRIPTION OF THE INVENTION

The present invention provides novel chemically inducible promoters andpromoter systems, its modification for use in plants (or other types ofcells, including animal cells), and its use in plants (and otherorganisms) to control gene expression, where the inducing chemical isnon-toxic. An exemplary inducible promoter was isolated from Aspergillusniger. Specifically, the novel chemically inducible promoter andpromoter system identified in Aspergillus niger is induced by benzoateand related compounds.

The following section describes the development of the invention,including the discovery of the benzoate-inducible fungal promoter andpromoter system, modification of the fungal promoter system and use inconstruction of gene promoter systems, and use of the hybrid fungal/genepromoter systems to control gene expression and developmental phases.Although the design of the promoter system uses plants as an example,the promoter system can be incorporated into, for example, animal cellsystems and other fungal systems that lack benzoate-activated promoters.

The resulting benzoate inducible fungal promoter and promoter system ofthe present invention is of general utility for controlling geneexpression in response to benzoic acid. It is understood that the methodof discovering the benzoate inducible promoter and promoter system,outlined above and described below in detail for Aspergillus niger, canbe applied to discover additional chemically inducible promoters fromAspergillus niger, as well as to discover chemically induciblepromoters, and in particular benzoate inducible promoters, from otherfungi or other organisms (e.g. plants).

I. Benzoate-Inducible Promoter, Response Element, Hybrid Promoters, andPromoter System

The present invention provides novel benzoate inducible promoters,promoter response elements, and promoter systems. In some embodiments,the present invention provides an isolated nucleic acid sequence of abenzoate inducible promoter region, where the promoter region is inducedby the presence of benzoate and/or related chemicals. In particularembodiments, the present invention provides an isolated nucleic acidsequence of a benzoate inducible promoter region from a fungus; infurther particular embodiments, the benzoate inducible promoter regionis from Aspergillus niger. In other particular embodiments, the benzoateinducible promoter region is from a bphA gene; in further particularembodiments, the bphA gene is from Aspergillus niger. In furtherparticular embodiments, the present invention provides an isolatednucleic acid sequence of a benzoate inducible promoter region comprisingSEQ ID NO:6 (as shown in FIG. 1, where SEQ ID NO:6 is positions −531 to−1 of SEQ ID NO:1), and its functional equivalents. In yet otherparticular embodiments, the present invention provides an isolatednucleic acid sequence of a benzoate inducible promoter region comprisinga 0.4 kb nucleic acid sequence fragment (SEQ ID NO:2) which is positions−199 to −521 of SEQ ID NO:1. In yet other particular embodiments, thepresent invention provides an isolated nucleic acid sequence of abenzoate inducible promoter region comprising BREF51 (which is theBRE-containing Fragment, 51 bp long, SEQ ID NO: 4, and which comprisespositions −357 to −407 of SEQ ID NO:1). In yet other particularembodiments, the present invention provides an isolated nucleic acidsequence of a benzoate inducible promoter region comprising any nucleicacid sequence fragment of SEQ ID NO:6, which fragment is at least about51 base pairs in length and also comprises SEQ ID NO:4. In yet otherparticular embodiments, the present invention provides an isolatednucleic acid sequence of a benzoate inducible promoter region comprisingany nucleic acid sequence fragment of SEQ ID NO:6 which is at leastabout 20 base pairs in length and which also comprises at least one BRE(for example, BRE6, SEQ ID NO:5 or BRE9, SEQ ID NO:8 which has thesequence CATTAGTCA).

In other embodiments, the present invention provides an isolated nucleicacid sequence comprising SEQ ID NO:6 (as shown in FIG. 1, where SEQ IDNO:6 is positions −531 to −1 of SEQ ID NO:1). In some embodiments, thepresent invention provides an isolated nucleic acid sequence comprisingBREF51 (which is the BRE-containing Fragment, 51 bp long, SEQ ID NO:4).In other embodiments, the present invention provides an isolated nucleicacid sequence of a benzoate response element (BRE6), which is thesequence of the 6 bp direct repeats of BREF51, with the sequence TAGTCA(SEQ ID NO:5). In other embodiments, the present invention provides anisolated nucleic acid sequence of a benzoate response element (BRE9),which is the sequence of a 9 nucleic acid sequence of the putative BRE9,with the sequence CATTAGTCA (SEQ ID NO:8). In other embodiments, severalcopies (e.g. 2-15) of BRE6 (SEQ ID NO:5) are incorporated into flankingsequences that provide suitable conformational and enhancement ofpromoter activity in a benzoate-specific manner.

The discovery of the promoter region, promoter, promoter responseelement, and fragments comprising the promoter response element isdescribed below. Further refinement of EMSA showed that a 51-bp sequencewithin mini-fragment FR6, at positions −357 to −407 from thetranscription start point, is a target-binding site of the protein(s).This sequence was then called BREF51 (highlighted in FIG. 1). Asignificant feature of this element is the presence of a pair of 6-bpdirect repeats, with the sequence 5′TAGTCA3′ (SEQ ID NO:5), which arelocated at the edges of the sequence. Direct repeats have been shown toconstitute target-binding sites of several different kinds oftranscription factors in a variety of species (Lee et al. (1995) Mol.Cell. Biol. 15: 4194-4207; Zelhof et al. (1995) Proc. Natl. Acad. Sci.USA 92: 10477-10481; Yin et al. (1997) Plant J. 12: 1179-1188;González-Pérez et al. (1999) J. Biol. Chem. 274: 2286-2290; Park et al.(1999) FEBS Letters 463: 133-138; Yu et at. (2000) J. Biol. Chem. 275:24208-24214; Risoen et al. (2001) Mol. Gen. Genom. 265: 198-206). Inmost instances these factors bind the target site as homodimers, withthe spacing between the repeats being essential for the proper bindingand function.

To confirm that the protein(s) previously shown to interact with BREF51do so by binding to one or more of the direct repeats, mutations wereintroduced on both repeated sequences, and these modified BREF51 werelabeled and used on EMSA. FIG. 10 shows that sequence modifications ofthe first repeat (position −407) do not interfere with the in vitrobinding of the protein factor(s). However, sequence modifications orcomplete substitution of the second TAGTCA (position −365 bp) repeatalmost completely abolishes binding, as judged by the lack of mobilityshift of the probe. Therefore, these results indicate that this (these)protein(s) is (are) binding specifically to the TAGTCA sequence locatedat −365 bp upstream of the transcription start point, within BREF51.However, since a faint mobility-shifted band was still observed when themodified probes were used, it is possible that sequences flanking theTAGTCA located at position −365 bp may also contribute to binding, orthere is weak binding to the upstream TAGTCA. Of special interest is thepresence of an almost perfect palindromic sequence (boxed in FIG. 10)positioned between the direct repeats. As shown in FIG. 10, the TGAsequence located just downstream from the second TAGTCA (position −365)does not seem to be important for this interaction. The promoter regionof the bzuA gene from A. nidulans also contains a TAGTCA sequence, whichis located −568 to −573 bp upstream from the transcription start point.However, this sequence neither appears to be duplicated in the promoterof that gene, nor does it contain a similar palindrome nearby the TAGTCAsequence.

These modified BREF51 sequences (or other candidate BREs) can beintroduced upstream of a “minimal promoter” (−331 to +1), and fused tothe GFP gene to further test these constructs. These constructs may betransformed into A. nidulans, and GFP expression scored in the presenceand absence of benzoic acid.

In other embodiments, the present invention provides nucleic acidsequences that hybridize to a benzoate inducible promoter region (forexample, SEQ ID NO:6), to a response element (for example, SEQ ID NO:4or SEQ ID NO:5), or to fragment comprising a benzoate response element(for example, SEQ ID NO:3), any and all as described above. In furtherembodiments, these sequences are functionally equivalent to benzoateinducible promoter, to a response element, or to a fragment comprisingthe benzoate response element, any and all as described above. Byfunctional equivalence, it is meant that these nucleic acid sequencesare capable of induction by benzoate and/or related chemicals either bythemselves, or when incorporated into a hybrid promoter (as describedfurther below), and when operably linked to a gene of interest. Suchsequences of the present invention are characterized for functionalequivalence using the methods described below. In other words, theprotocols and examples below may be used to test any of the sequencesfound to hybridize to the sequences described above to determine if theywill function as benzoate response elements (e.g. in combination with agene promoter).

In other embodiments, the present invention provides nucleic acidsequences of benzoate inducible promoters, response elements, andpromoter fragments comprising response elements, which sequences arenaturally located upstream to structural DNA sequences which areidentified as genes naturally under control of a homologous benzoateinducible promoter as described above, where any of the naturallyoccurring nucleic acid sequences are functionally equivalent to thebenzoate inducible promoters, response elements, and promoter fragmentscomprising response elements as described above.

In other embodiments, the present invention provides a novel benzoateinducible hybrid promoter, where the hybrid promoter comprises at leastone copy of any nucleic acid sequence fragment of SEQ ID NO:6, whichfragment is at least about 20, about 30 or about 51 base pairs in lengthand also comprises SEQ ID NO:5, and a heterologous gene promoter region,such that the hybrid promoter is responsive to benzoate and/or relatedchemical inducers. In particular embodiments, the nucleic acid sequencefragment of SEQ ID NO:6 is BREF51 (SEQ ID NO:4) or any other fragmentthat contains BRE6 (SEQ ID NO:5) and is at least 15 or at least 20 basesin length. In other embodiments, the present invention provides a novelbenzoate inducible hybrid promoter, where the hybrid promoter comprisesat least one copy of any nucleic acid sequence fragment of SEQ ID NO:6,which fragment comprises at least one copy of a BRE (for example, BRE6,SEQ ID NO:5, or BRE9, SEQ ID NO:8), and a heterologous gene promoterregion, such that the hybrid promoter is responsive to benzoate and/orrelated chemical inducers (e.g. benzoate mimetics, which may be foundusing the protocols described in the examples below by substituting acandidate mimentic for benzoate). In other embodiments, the presentinvention provides a novel benzoate inducible hybrid promoter, where thehybrid promoter comprises at least one copy of a BRE (for example, BRE6,SEQ ID NO:5, or BRE9, SEQ ID NO:8) and a heterologous gene promoterregion, such that the hybrid promoter is responsive to benzoate and/orrelated chemical inducers. In any of these embodiments, the heterologousgene regulatory region may further comprise minimal gene promoterregions and/or elements. In particular embodiments, a minimal plantpromoter comprises a TATA box for RNA polymerase recognition but noother elements that give cell or tissue specificity. The BREs in thesehybrid promoters are believed to be bound by protein factors, which areencoded by constitutively expressed transcriptional factor genes, andwhich modulate promoter activity in the presence of benzoate and/orrelated chemical inducers. As described below, optimization of hybridpromoter constructs involves determining the minimal fragmentscomprising at least one BRE necessary and sufficient to control geneexpression. In some embodiments, tandem arrays of BREF51 are linked tominimal promoter regions sufficient to create (BREF51)_(n)-minimalpromoters that are utilized in host cell systems; in some furtherembodiments, the minimal promoter region is a plant minimal promoterregion, and the hybrid promoter is utilized in plant systems. Generegulatory regions are derived from eukaryotic or prokaryotic cells;eukaryotic cells are plant or animal cells. Such novel benzoateinducible hybrid promoters of the present invention are characterizedfor functional equivalence using the methods described below.

In other embodiments, the present invention provides a novel benzoateinducible promoter system, which comprises a promoter component and atleast one transacting factor component. The promoter component comprisesany of the benzoate inducible promoters of the present inventiondescribed above. The transacting factor component comprises a codingregion for a transacting factor that is necessary and sufficient for theinduction of the promoter component by benzoate and/or relatedchemicals; in some embodiments, the transacting factor is atranscription factor. In some embodiments, the at least one additionalgene encoding a transacting factor is under control of a promoter; insome embodiments, the promoter is a constitutive promoter. In someembodiments, the benzoate promoter system is present in at least oneexpression vector, where the expression vector(s) can be used totransfect a host cell, either transiently or stably. Discovery oftranscription factor components, and further description of thesecomponents, is provided below.

Briefly, the yeast one-hybrid system works by construction of a libraryof many thousands of clones of potential transcription factors in yeastclones in which three tandem copies of BREF51 (SEQ ID NO:4) driving agene that would enable the yeast to grow on media lacking the amino acidHis. The yeast one-hybrid system screen yielded 17 colonies onSD/-Leu/-His media after 4 days of incubation. These 17 clones werestreaked on SD/-Leu/-His media to confirm the ability of the cells togrow in the absence of the two amino acids. Plasmid DNA obtained fromall the clones was used as a template for PCR reactions with the purposeof determining the size of the cDNA inserts. The inserts varied in sizefrom 0.5 to 1.4 kbp. Some clones contained more than one insert. PlasmidDNA from the positive clones was transformed into E. coli XL1-Blue cellswith the objective of obtaining greater amounts of DNA for sequencing,as well as to be able to separate distinct plasmids contained within thesame yeast strain. Plasmid DNA was prepared from the bacterial clonesand used as template for a new round of PCR. The results from thesereactions confirmed that some of the original yeast clones indeedcontained two distinct plasmids (data not shown). In contrast to E.coli, yeast cells are able to maintain and replicate more than oneplasmid inside the cell. DNA sequences obtained from the plasmidsisolated from the bacterial cells were translated and compared by BLASTpsearch (Altschul et al. (1997) Nucl. Acids Res. 25: 3389-3402) to theGenBank database (Table 1).

TABLE 1 Sequence similarity of GAD clones to available databases (BLASTpsearch). CLONE INSERT SCORE ID SIZE SIMILARITY (bits) E VALUE GAD 1.11.2 kbp Hypothetical prot. 115 7e⁻²⁵ N. crassa, serine-rich prot. 821e⁻¹⁴ S. pombe GAD 2 NO INSERT GAD 3.1 1.2 kbp Pepsinogen A. niger 583 e⁻¹⁶⁵ GAD 3.2 1.3 kbp Hypothetical prot. 243 2e⁻¹⁹ N. crassa GAD 4.11.0 kbp hypothetical prot. 104 2e⁻²¹ N. crassa, CipC protein 77 5e⁻¹³ A.nidulans GAD 4.2 0.6 kbp No significant — — similarities GAD 5.1 1.2 kbphypothetical prot. 104 2e⁻²¹ N. crassa, CipC protein 77 5e⁻¹³ A.nidulans GAD 6.1 1.1 kbp Isopentenyl diphosphate 103 4e⁻²¹ isomerase A.nidulans GAD 6.2 0.8 kbp 60s ribosomal protein 215 7e⁻⁵⁵ S. pombe GAD7.1 0.7 kbp No significant — — similarities GAD 8.1 0.6 kbp Predictedprot. 68 2e⁻¹⁰ N. crassa GAD 9.1 0.5 kbp No significant — — similaritiesGAD 10.1 0.8 kbp Hypothetical prot. 171 1e⁻⁴¹ N. crassa, putativebacterial 161 1e⁻³⁸ flavohemoprotein GAD 11.1 1.0 kbp No significant — —similarities GAD 12.3 1.0 kbp Triose phosphate 429  e⁻¹¹⁹ isomerase A.niger GAD 12.4 1.2 kbp Outer mitochondrial 374  e⁻¹⁰⁵ membrane prot.porin N. crassa, yeast GAD 13.1 0.6 kbp No significant — — similaritiesGAD 14.1 0.5 kbp No significant — — similarities GAD 15 NO INSERT GAD16.1 0.6 kbp Hypothetical prot. 93 6e⁻¹⁸ B17C10.140 N. crassa GAD 16.31.3 kbp Hypothetical prot. 105 1e⁻²¹ N. crassa, Hypothetical prot. 1033e⁻²¹ S. pombe GAD 17.1 1.2 kbp Heat shock prot. 457  e⁻¹²⁷ 70 A.nidulans

Some clones, such as GAD3.1, GAD12.3, GAD12.4, and GAD17.1 constitutedfalse positives, in the sense that the proteins had very high similarityto fungal proteins which functions are known not to be involved withtranscriptional regulation. However, most of the cDNAs encoded proteinswhose functions are not yet known. These are mostly hypotheticalproteins predicted from computer algorithms, and generated during datamining of complete genome sequencing projects.

Translated sequences of potential candidates for proteins that interactwith BREF51 were also compared to completed genome sequences ofAspergillus nidulans (Release 1) and Neurospora crassa (Release 3)(tBLASTn search, Whitehead Institute) (Table 2), since both of thesespecies contain a gene with high similarity to the bphA gene from A.niger.

TABLE 2 Comparison of selected GAD sequences to A. nidulans and N.crassa completed genome sequences (tBLASTn search). CLONE SIMILARITYSCORE SIMILARITY SCORE ID TO A. nidulans (E value) TO N. crassa (Evalue) GAD 1.1 Contig 1.64 306 (2e⁻⁸³) Contig 3.74 119 (4e⁻²⁷) GAD 3.2Contig 1.14 359 (2e⁻⁹⁹) Contig 3.190  94 (3e⁻¹⁹) GAD 4.1 No hits — Nohits — GAD 4.2 Contig 1.51  70 (1e⁻¹²) No hits — GAD 5.1 No hits — Nohits — GAD 7.1 No hits — No hits — GAD 8.1 No hits — No hits — GAD 9.1No hits — No hits — GAD 10.1 Contig 1.122 256 (1e⁻⁶⁸) Contig 3.94 167(6e⁻⁴²) GAD 11.1 Contig 1.69 124 (6e⁻²⁹) No hits — GAD 13.1 No hits — Nohits — GAD 14.1 Contig 1.105 144 (2e⁻³⁵) No hits — GAD 16.1 Contig 1.174 86 (1e⁻¹⁷) Contig 3.75  67 (7e⁻¹²) GAD 16.3 Contig 1.19 147 (6e⁻³⁶)Contig 3.210 105 (6e⁻²³)

As demonstrated by RNA blots, the A. nidulans homolog bzuA is alsoupregulated in the presence of benzoic acid in the media, and despiteshowing a different pattern of expression, it is likely that A. nidulanscontains a similar machinery to control bzuA activity. On the otherhand, while N. crassa also contains a bphA homolog, there is no currentinformation available as to its response to benzoic acid.

Total proteins were isolated from 9 yeast clones which cDNAs encodedproteins that did not show significant similarities to any gene of knownfunction in the databases. Protein extracts from each of these yeastclones were used in EMSA to eliminate those cDNAs that encode proteinsthat do not interact with BREF51. Yeast clones GAD1 (amino acid sequenceshown in SEQ ID NO:15; and nucleic acid sequence shown in SEQ ID NO:16)and GAD11 (amino acid sequence shown in SEQ ID NO:17; and nucleic acidsequence shown in SEQ ID NO:18), which contained only one cDNA each, asjudged by PCR data, both showed a gel mobility shift of the BREF51fragment in these assays (FIG. 13). The shift in mobility displayed byGAD1 was similar to that observed with A. niger total protein extracts.The protein encoded by GAD1 (SEQ ID NO:15) is highly similar to apredicted protein present in the A. nidulans genome (E value 2e⁻⁸³), andsimilar to a hypothetical protein (B24P11.210) in the N. crassa genome(E value 4e⁻²⁷) (Table 2). GAD1 protein is rich in serine residues(16%), and is predicted by PSORTII (Nakai and Horton (1999) TrendsBiochem. Sci. 24: 34-35) to be localized in the nucleus (70.6%probability). It also shows high similarity to a hypotheticalserine-rich protein (C13G6.10c) from S. pombe. Serine-rich motifs can bephosphorylation targets by protein kinase C in proteins (Branden andTooze (1999) Introduction to protein structure, 2^(nd) ed. GarlandPublishing, New York, N.Y. 410 p.). Also, serine-rich regions have beenshown to be part of the transactivation domains of some transcriptionfactors in both mammalian and viral cells (Wetering et al. (1993) EMBO.J. 12: 3847-3854; Ma and Staudt, (1996) Blood 15: 734-745; Chen et al.(1999) Mol. Cell. Biol. 19: 307-316; Bowles et al. (2000) J. Virol. 74:1200-1208). While not necessary to understand in order to practice thepresent invention, if GAD1 is involved in the regulation of BPHprom,GAD1 protein might be always bound to a benzoic acid response element inthe bphA promoter. In the absence of benzoic acid GAD1 would beinactive, unable to promote transcription. However, addition of benzoicacid could cause phosphorylation of one of the serine residues in GAD1,leading to its activation. This mode of regulation is analogous to thatof the transcription factor CREB, which is involved in responses to cAMPin human cells (Latchman (1997) Intl. J. Biochem. Cell Biol. 29:1305-1312).

GAD11 protein (SEQ ID NO: 17) is weakly similar to a Drosophilamelanogaster homeotic gene regulator, and also to a putative nuclearprotein family member from the nematode C. elegans. It is also predictedby PSORTII (Nakai and Horton (1999) Trends Biochem. Sci. 24: 34-35) tobe localized in the nucleus (94.1% probability). GAD11 was alsopredicted to have a coiled-coil region consisting of 37 amino acidresidues (Lupa's algorithm). The α-helical coiled coil is a structuralmotif found in many proteins. It consists of two long α-helices with arepeating pattern of hydrophobic side chains that interlock to produce asupercoiled structure. The most important function of coiled-coilregions in myosins and kinesins is for dimerization. Because the coiledcoil may be rigid and extended in solution it can act as a spacer orconnector between protein domains (Branden and Tooze (1999) Introductionto protein structure, 2^(nd) ed. Garland Publishing, New York, N.Y. 410p.). While not necessary to understand to practice the presentinvention, this may explain the larger shift in BREF51 mobility in thepresence of GAD11 protein extract, since GAD11 proteins may be forminghomodimers connected by the coiled-coil region. GAD11 has a homolog inA. nidulans (E value 6e⁻²⁹), but no homologs in N. crassa were found.Although 5 μg of total yeast protein extracts were used on EMSA, the gelmobility shifts observed on the yeast clones were less intense thanthose obtained with the positive control, where only 1 μg of totalprotein extract from A. niger cells was used. According to the plasmidmanufacturer (Clontech), cDNAs cloned into this activation domain vectorshow low expression levels, which may explain the lower intensity of theshifted bands even in the presence of a higher amount of total proteins.

Reverse Transcription PCR (RT-PCR) experiments revealed that while GAD1is expressed constitutively, GAD11 is slightly downregulated in thepresence of 8 mM benzoic acid for 5 h (FIG. 14). While not necessary tounderstand to practice the present invention, if in fact either GAD1 orGAD11 are involved in regulation of the bphA promoter, constitutivetranscription of GAD1 does not rule out the possibility that theactivity of the GAD1 protein is modulated by benzoic acid. On the otherhand, downregulation of the GAD11 gene in the presence of benzoic acidindicates that the protein may function as a transcriptional repressorof the bphA promoter. GAD1 is constitutively expressed, while GAD11seems to be downregulated in the presence of benzoic acid in the media.There is a possibility that protein-protein interactions play a role inthe regulation of this gene in A. niger. To clarify the role of GAD1 andGAD11, one could create deletion mutations of each gene (GAD1 and GAD11)in A. niger. Deletion of the true transcription factor that controlsactivity of BPHprom should result in a lack of bphA expression in thepresence of benzoic acid. Alternatively, deletion mutants of the GAD1and GAD11 homologs from A. nidulans strains containing the pBPH-1847-GFPconstructs could be prepared, and GFP expression in the presence ofbenzoic acid is scored.

Benzoate inducible means that the promoter is inducible by benzoateand/or related chemicals. Benzoate and/or related chemicals include butare not limited to benzoic acid and its salts (Na⁺, K⁺, NH₄ ⁺, etc.),and esterified forms of benzoic acid. The esterified forms of benzoicfacilitate penetration of the esterified forms into cells, and onceinside the cells, the esterified forms are subsequently be de-esterifiedby the cell's own esterases to yield active compound. Thus, relatedchemicals include but are not limited to sodium benzoate and methylbenzoate. It is contemplated that features necessary and sufficient foractivity are a benzyl ring with one carboxyl group. It is contemplatedthat derivatives with substitutions at the ortho- (e.g.,2-hydroxy-benzoate, or salicylate) and meta-positions of the ring reduceor abolish effectiveness of the benzoic acid derivative as an inducer,whereas substitutions at the para-position, such as para-aminobenzoate,are generally more effective.

Discovery of the benzoate inducible promoter and promoter responseelement involved several steps. These included identifying a gene whichis induced by benzoic acid, characterizing induction of expression,isolating the promoter region, determining which fragments of thepromoter region comprise a benzoate response element, and identifyingadditional components of the benzoate inducible promoter system, such astransacting factors, where such transacting factors include but are notlimited to transcription factors. Modification of the benzoate induciblepromoter system includes but is not limited to incorporating thebenzoate response elements into a gene promoter or regulatory region tocreate a benzoate inducible hybrid promoter, and in particular into agene minimal promoter or regulatory region. Utilization of the benzoatepromoter system includes but is not limited to transforming cells withthe benzoate inducible hybrid promoter; in some embodiments, utilizationfurther includes the additional transformation of cells with at leastone gene encoding an additional factor which allows the benzoateinducible hybrid promoter to function in the transgenic cell and beinducible by benzoate and/or related chemicals; such factors includetranscription factors. The greater the disparity between the source ofthe benzoate inducible promoter and the host cell (such as a fungus anda plant, respectively), the greater the likely disparity betweeninducible factors between the two types of cells. Thus, it iscontemplated that a greater specificity of the inducible factors isobtained in an inter-Kingdom combinatorial system, which means that thesource inducing factors are less likely are to induce endogenous genesin the host cell.

A. Discovery of Benzoate-Inducible Promoter and Promoter ResponseElement

As reviewed by Gatz et al. ((1997) Ann. Rev. Plant Physiol. Plant Mol.Biol. 48: 89-108) and Gatz and Lenk ((1998) Trends Plant Sci. 3:352-358.), an ideal chemically inducible expression system would possessall of the following features: a) expression levels should be very lowor none in the absence of the chemical stimulus; b) expression levelsshould increase rapidly to high levels upon application of the inducer;c) the chemical stimulus should be non-toxic to the plant and all otherorganisms in the plant's ecosystem; d) the chemical stimulus should notexhibit pleiotropic effects in treated plants; e) the chemical stimulusshould be easily applied in the field and in the greenhouse by spraying,or under tissue culture conditions by adding it to a synthetic medium;f) depending on the nature of the application, different derivatives ofthe inducer should be available and induction should be effective at lowrates of application; g) a second compound should be available thatabrogates the induction so that both positive and negative control arepossible; h) a chemically inducible system should also be amenable to becombined with tissue-specific expression.

Several examples of chemical control of gene expression in plants existtoday, but none meets all of the requirements described above. Theseexamples include synthetic promoter systems that can be induced bytetracycline (Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Shockett and Schatz (1996) Proc. Natl. Acad. Sci. USA 93,5173-5176), steroid compounds (Aoyama and Chua (1997) Plant J. 11:605-612; Kunkel et al. (1999) Nature Biotech. 17: 916-919), copper ions(Mett et al. (1993) Proc. Natl. Acad. Sci. USA 90: 4567-4571), ethanol(Caddick et al. (1998) Nature Biotech. 16: 177-180; Salter et al. (1998)Plant J. 16: 127-132), herbicide safeners (De Veylder et al. (1997)Plant Cell Physiol. 38: 568-577), and insect ecdysone agonists (Jepsonet al. (1998) Pestic. Sci. 54: 360-367). Each system has its owndisadvantages, but all share a major disadvantage of the high level oftoxicity of the inducers, especially if the inducers were to be used onfield crops. For example, growth defects have been reported intransgenic Arabidopsis plants containing the glucocorticoid-inducibletranscription system (Kang et al. (1999) Plant J. 20: 127-133). Otherdisadvantages are encountered using as chemical inducers chemicalsendogenously produced by the host system. As an example, plants makeethanol under oxygen stress, for example under flooding stress, so useof the ethanol promoter in agronomic settings has severe disadvantageswith respect to tightness of control. Thus, there are inherentadvantages to the use of a chemical not produced by the host organism inwhich the novel system has been placed.

The use of transcriptional activators and their corresponding responseelements from fungal systems is attractive because of the reduced riskof interference between the switch system and the endogenous host-planttranscriptional machinery (Jepson et al. (1998) Pestic. Sci. 54:360-367). Thus, for example, if the construction of a chemicallyinducible promoter is based on a plant gene, then application of thechemical inducer would also cause induction of the endogenous plantgene. The disparity between fungal and plant systems suggests thatfungal effectors would not be found in the plants, and thus employingfungal effectors minimizes any possible interference. One example is theethanol-controllable system mentioned above; this system is based uponthe Alc regulon from the fungus Aspergillus nidulans (Eidam) Winter,which controls its response to ethanol and other related chemicals. Thissystem has been only recently developed (Caddick et al. (1998) NatureBiotech. 16: 177-180), and, although it seems initially promising,questions concerning the specificity of the inducer for the transgene,toxicity, background levels, and induction levels over a longer timecourse have yet to be addressed (Gatz and Lenk (1998) Trends Plant Sci.3: 352-358). Therefore, other fungal promoters may be more suitable, oroffer other advantages.

The fungus Aspergillus niger appeared to be a likely source of asuitable promoter system based upon the following observations.Aspergillus niger is able to grow in a culture medium that contains onlybenzoic acid as a carbon source, as reported in 1985 by Sahasrabudhe andModi ((1985) Biochem. Intl. 10: 525-529). The isolation and cloning ofthe gene that encodes the enzyme benzoate para-hydroxylase (BPH) fromAspergillus niger (BphA gene; GenBank X52521) was reported in 1990 byvan Gorcom et al. ((1990) Mol. Gen. Genet. 223, 192-197). BPH catalyzesthe first of a series of steps by which A. niger catabolizes benzoicacid; this first step involves the hydroxylation of the aromatic ring atthe para position. The enzyme belongs to the class of cytochrome P450monooxygenases (CYP53A1; van den Brink et al. (1998) Fungal Genet. Biol.23, 1-17) and was first purified by Reddy and Vaidyanathan (1975)Biophys. Acta 384: 46-57). The BPH enzyme is not present in the fungalmycelia if benzoate is not added to the culture medium, and itssynthesis seems to be transcriptionally induced upon transfer of thefungal cells to benzoate-containing media (van Gorcom et al. (1990) Mol.Gen. Genet. 223: 192-197). Hence, this gene appeared to be a goodcandidate for gene induction at the transcriptional level by aninnocuous chemical substance.

1. Identification of the Promoter

Investigation of the distribution of the gene encoding the enzymebenzoate para-hydroxylase (BPH) in plants and fungi demonstrated thatthe bphA gene is found only in fungi (FIG. 2). Investigation of the timeof gene induction indicated that the bphA gene is induced by benzoicacid after only ten minutes of exposure to the chemical (FIG. 3). Thepattern of expression of the bphA gene from A. niger was investigatedwith a series of RNA blots; the results indicate that the bphA genepromoter is quickly induced within 10 minutes after incubation of themycelia in media containing 8 mM benzoic acid (FIG. 3). Concentrationcurves indicate that benzoic acid concentrations as low as 0.8 mM inducetranscription of the bphA gene. Other compounds which can induce thepromoter include sodium benzoate and methyl benzoate; however, benzylalcohol and hydrocinnamic acid do not induce the promoter (FIG. 4). Inall of these RNA blot experiments, it was observed that the BPH messageis not present until benzoate is added to the culture medium. Therefore,it is contemplated that this gene might provide some strict regulatorymechanisms for control of gene expression.

Using Anchored Polymerase Chain Reactions (PCR) as described by (Siebertet al. (1995) Nucl. Acids Res. 23: 1087-1088), about 1.8 kb of thepromoter region of the Aspergillus niger bphA gene was isolated, andthen cloned into pBluescript SK⁻. This region was subsequently sequencedby an automated di-deoxy chain termination method (Sanger et al. (1977)Proc. Natl. Acad. Sci. USA 74 5463-5467) at a commercial nationalservice center; the sequence is shown in FIG. 1.

2. Identification of the Benzoate Response Element

In order to investigate whether protein factors are involved incontrolling the initiation of transcription from the bphA gene promoter,seven mini-promoter fragments were created by PCR (FIG. 5), ranging insize from 247 bp to 324 bp. These fragments were then radio-labeled with³²P-dATP, and used in Electrophoretic Mobility-Shift Assays (EMSA).After confirming that one of these fragments (FR6, 323 bp) was bound byprotein factors (FIG. 6), partially overlapping sub-fragmentsencompassing the FR6 fragment were prepared, and again used in EMSA.These assays showed that a 51-bp fragment located 350 bp upstream of thetranscription start point is consistently bound by a protein factor thatis present in total protein extracts from benzoate-induced A. nigermycelia (FIG. 7). The most significant feature of the sequence of this51 bp fragment (shown in FIG. 8) is the presence of a pair of 6-bpdirect repeats (underlined in FIG. 8). Short direct repeats have beenshown to constitute cis-acting promoter elements affecting transcriptionof many genes (Lee et al. (1995) Mol. Cell. Biol. 15: 4194-4207; Zelhofet al. (1995) Proc. Natl. Acad. Sci. USA 92: 10477-10481; Yin et al.(1997) Plant J. 12: 1179-1188; González-Pérez et al. (1999) J. Biol.Chem. 274: 2286-2290; Park et al. (1999) FEBS Lett. 463: 133-138; Yu etal. (2000) J. Biol. Chem. 275: 24208-24214; and Risoen et al. (2001)Mol. Gen. Genom. 265: 198-206). These repeats are usually bound bytranscription factors that form dimers when in the active form.Therefore, the number of nucleotide bases between the direct repeats isoften critical for achieving binding of the protein factor. However, asdiscussed above, the functioning BRE may comprise only one of therepeats. In fact, it was found that all or most of the activity wasretained by employing only one of the BRE6 repeats. The single consensuselement found in the bphA promoters of A. niger, A. nidulans, and N.crassa is TAGTCA (SEQ ID NO:5). It is contemplated that, in someembodiments, nucleotide bases flanking this putative element areincluded for complete binding of a transcription factor.

When mutations were introduced on both repeated sequences, and thesemodified BREF51 were labeled and used on EMSA, sequence modifications ofthe first repeat (upstream position −407) do not interfere with the invitro binding of the protein factor(s). However, sequence modificationsor complete substitution of the second TAGTCA (downstream position −365bp) repeat almost completely abolishes binding, as judged by the lack ofmobility shift of the probe (FIG. 10). Therefore, these results indicatethat this (these) protein(s) is (are) binding specifically to the TAGTCAsequence located at −365 bp upstream of the transcription start point,within BREF51. However, since a faint mobility-shifted band was stillobserved when the modified probes were used, it is possible thatsequences flanking the TAGTCA located at position −365 bp are alsoimportant for binding, or there is weak binding to the upstream TAGTCA.

bphA gene promoter fusions to the Green Fluorescent Protein (GFP) genewere constructed and transformed into Aspergillus nidulans strain GR5(ATCC #200171). Both the full promoter region (1.8 kbp) and a shortversion containing fragment 6 and the TATA box (FR6, 0.4 kbp) wereplaced in front of the GFP coding region (pEBFP, Clontech, Palo Alto,Calif.). Transformants were screened by PCR for the presence of the GFPgene, and grown either in the presence or in the absence of 8 mM benzoicacid. After 5 hours of induction, both full-length and fragment 6 aloneof the promoter region were able to induce GFP expression only in thepresence of the inducer benzoate (FIG. 9).

Although a putative Benzoate Response Element (BRE) has been reported tobe located about 1 kb upstream from the translation start point (van denBrink et al. (2000) Fungal Genet. Biol. 23, 1-17), the results fromElectrophoretic Mobility Shift Assays (EMSA) described above showed thata 51 bp region located downstream from the reported BRE consistentlybinds to a factor present in a total protein extract frombenzoate-induced mycelia (FIG. 11). As described above, this 51 bpregion possesses a pair of 6-bp direct repeats, with the second onebeing necessary and sufficient for binding of the protein from the totalextract. Therefore, it is contemplated that this 51 bp fragment containsthe true BRE in the BphA promoter, and that the TAGTCA within the 51 bpfragment is the true BRE; the 51 bp fragment is referred to as the BREcontaining fragment of 51 nucleotides in length, or BREF51. Moreover,the putative BRE proposed by van den Brink et al. ((2000) Mol. Gen.Genet. 263: 601-609) lies in a region which encompasses a well conservedOpen Reading Frame (ORF) among fungal species, with no specific functionattributed to it to date; this ORF ends at position −532 of SEQ ID NO:1.

The identity of effective BRE(s), along with the minimal fragmentsize(s) and sequences for chemical inducibility by benzoate and/orrelated chemicals, is examined by generating fragments and modificationof BREF51 (SEQ ID NO:4) by well known techniques; these fragments arethen operably linked to a marker gene, and the expression of the markergene in response to benzoate and/or related chemicals examined. In thisway, additional BREs may be identified using the methods and proceduresherein. For example, one may test a fragment of SEQ ID NO:6 that is atleast 20 nucleotides in length and which contains BRE6 TAGTCA (SEQ IDNO:5). Such fragments may contain sequences upstream of BRE6 (the onelocated at −365 to −370) or downstream, or both upstream and downstream.Exemplary procedures are described above (and below in the Examples),where Green Fluorescent Protein (GFP) gene constructs are operablylinked to test promoters and transformed into Aspergillus nidulansstrain GR5 (ATCC #200171). The test promoter regions are placed in frontof the GFP coding region (pEBFP, Clontech, Palo Alto, Calif.), andtransformants screened by PCR for the presence of the GFP gene, andgrown either in the presence or in the absence of benzoic acid (at, forexample, about 8 mM). The appearance of GFP when A. nidulans is grown inthe presence of benzoic acid indicate that a test promoter regionfunctions effectively as a benzoate inducible promoter.

The 51 bp fragment (BREF51), and any subfragments or modifications ofthis region which function as benzoate inducible promoters, are used togenerate tandem arrays and combined with a gene promoter, including butnot limited to a plant gene promoter.

Thus, discovery of the response element is the first step in the designof a chemically inducible promoter, as described further below.

B. Identification and Cloning of the Gene(s) That Encode TFs That Bindto the BRE Regions Upon Induction by Benzoic Acid

While it is not necessary to understand the mechanism to practice thepresent invention, it is believe that that the mechanism of induction islikely to involve a soluble transcription factor (TF), such as GAD1 andGAD11, or similar proteins (See, e.g., FIGS. 16-19). Two generalmechanisms of increasing complexity by which benzoate induces thispromoter are depicted in FIG. 12. Regardless of the mechanism ofbenzoate perception, it is first established whether the transcriptionalcontrol is obtained through activation or depression. While notnecessary to understand or practice the present invention, it may benecessary not only to identify TFs that would bind to this promoter andregulate transcription of the bphA gene, but also to identify a membranesurface receptor that releases the TFs. A direct binding of benzoate tothe promoter is ruled out, in some embodiments, for the simple reasonthat no such mechanism of transcriptional regulation has ever beenidentified. In other embodiments, benzoate does directly bind the BRE.Control plants transformed with the promoter elements alone provideconfirmation of that assessment, or in the alternative indicate thatArabidopsis has its own benzoate inducible TF for some unidentifiedgene.

1. Internal Induction by Benzoate

FIGS. 12A and 12B depict binding of benzoate to a TF mediating eitheractivation or depression of the promoter. This hypothesis is testedfirst. Both a biochemical and a molecular affinity binding approach areused to identify the TFs. The binding of TFs in a benzoate-specificmanner is tested first; initial results are described below. Theseexperiments consist of electrophoretic mobility-shift assays (EMSAs) asdescribed by Singh et al. ((1986) Nature 319: 154-158). The region ofthe gel showing the mobility shift is excised, and the protein(s) boundto the DNA fragment are electroeluted. The purified protein(s) aresubjected to microsequencing, providing enough information for theidentification and cloning of the respective genes. Nuclei are isolatedfrom control or benzoate-induced A. niger mycelia according toestablished protocols (Richardson et al. (1992) Mol. Cell Biol. 12:337-346), and protein extracts containing the TFs are prepared. The BREis used to capture the TFs that are revealed by EMSA in abenzoate-specific manner.

Initial efforts to isolate trans-acting factor(s) involved in theregulation of the bphA gene promoter utilized affinity chromatographyexperiments using either the BRE51 or benzoic acid as affinity ligands.In a first approach, BRE was biotinylated, purified, and allowed to bindto streptavidin-linked paramagnetic particles (PMPs; PolyATtract mRNAIsolation Kit, Promega, Madison, Wis.). Total protein extract frombenzoate-induced A. niger was added to the solution, incubated for 60min, and the PMPs were washed several times. Bound proteins were theneluted from the PMPs under high salt conditions. The fractions wereelectrophoresed on SDS-PAGE, and a protein of approximately 45 kDa couldbe observed (FIG. 11); however, this factor failed to cause a shift onthe mobility of BRE51 when used on EMSA. In a second approach, benzoicacid, immobilized on 4% cross-linked beaded agarose (Sigma, St. Louis,Mo.) was used as the affinity ligand. Again, total protein extract frombenzoate-induced A. niger was added, the column was washed, and boundproteins eluted under high salt conditions. The eluted fraction alsofailed to cause mobility shifts on labeled BRE51 when used on EMSA.

Calculation of the stoichiometry of the amount of binding of a proteinelement that saturates the amount of BREF51 in the EMSA indicates that aminimum of 6 fmoles are present per mg of protein extract. If the factoris about 50 kDa, then this amount represents 300 pg of factor. Thus, amanageable scale-up of about 100-fold is necessary to bind 30 ng offactor needed to detect tryptic fragments by MALDI-TOF MS.

To identify TFs that bind to the BRE, an alternative approach utilizes ayeast 1-hybrid system (Sieweke (2000) Meth. Mol. Biol. 30: 59-77).Simply, the promoter element to be used as “bait” is inserted into ayeast minimal promoter-HIS3 reporter (Clontech). The yeast cells, whichare unable to grow in the absence of histidine, are transformed withthis construct to create a reporter strain. This reporter strain is thentransformed with a GALA activation domain (AD)-cDNA fusion libraryprepared from A. niger. The transformants are plated out on minimalmedium lacking histidine. If the AD fusion protein interacts with thepromoter fusion, then the yeast will acquire the ability to grow onmedia without histidine. The plasmid containing the positive cDNA ispurified from this colony and sequenced. Such an approach often yieldsseveral false positives in addition to true factors. However, each ofthese recombinant proteins can be tested in a BRE-GFP system to confirmfunctionality in a timely manner.

As described in more detail earlier, the yeast one-hybrid system screenyielded 17 colonies on SD/-Leu/-His media. DNA sequences obtained fromthe plasmids isolated from the bacterial cells were translated andcompared by BLASTp search (Table 1). Some clones, such as GAD3.1,GAD12.3, GAD12.4, and GAD17.1 constituted false positives, in the sensethat the proteins had very high similarity to fungal proteins whichfunctions are known not to be involved with transcriptional regulation.However, most of the cDNAs encoded proteins whose functions are not yetknown. These are mostly hypothetical proteins predicted from computeralgorithms, and generated during data mining of complete genomesequencing projects. Translated sequences of potential candidates forproteins that interact with BREF51 were also compared to completedgenome sequences of Aspergillus nidulans (Release 1) and Neurosporacrassa (Release 3) (tBLASTn search, Whitehead Institute) (Table 2),since both of these species contain a gene with high similarity to thebphA gene from A. niger.

Total proteins were isolated from 9 yeast clones which cDNAs encodedproteins that did not show significant similarities to any gene of knownfunction in the databases. Protein extracts from each of these yeastclones were used in EMSA to eliminate those cDNAs that encode proteinsthat do not interact with BREF51. Yeast clones GAD1 (FIG. 16) and GAD11(FIG. 17), which contained only one cDNA each, as judged by PCR data,both showed a gel mobility shift of the BREF51 fragment in these assays(FIG. 13). The shift in mobility displayed by GAD1 was similar to thatobserved with A. niger total protein extracts. The protein encoded byGAD1 is highly similar to a predicted protein present in the A. nidulansgenome (E value 2e⁻⁸³), and similar to a hypothetical protein(B24P11.210) in the N. crassa genome (E value 4e⁻²⁷) (Table 2). GAD1protein is rich in serine residues (16%), and is predicted by PSORTII tobe localized in the nucleus (70.6% probability). It also shows highsimilarity to a hypothetical serine-rich protein (C13G6.10c) from S.pombe. Serine-rich motifs can be phosphorylation targets by proteinkinase C in proteins. Also, serine-rich regions have been shown to bepart of the transactivation domains of some transcription factors inboth mammalian and viral cells. Therefore, while not necessary tounderstand or practice the present invention if GAD1 is involved in theregulation of BPHprom, GAD1 protein might be always bound to a benzoicacid response element in the bphA promoter. In the absence of benzoicacid GAD1 would be inactive, unable to promote transcription. However,addition of benzoic acid could cause phosphorylation of one of theserine residues in GAD1, leading to its activation. This mode ofregulation is analogous to that of the transcription factor CREB, whichis involved in responses to cAMP in human cells (Latchman 1997).

GAD11 protein is weakly similar to a Drosophila melanogaster homeoticgene regulator, and also to a putative nuclear protein family memberfrom the nematode C. elegans. It is also predicted by PSORTII to belocalized in the nucleus (94.1% probability). GAD11 was also predictedto have a coiled-coil region consisting of 37 amino acid residues(Lupa's algorithm). The α-helical coiled coil is a structural motiffound in many proteins. It consists of two long α-helices with arepeating pattern of hydrophobic side chains that interlock to produce asupercoiled structure. The most important function of coiled-coilregions in myosins and kinesins is for dimerization. Because the coiledcoil may be rigid and extended in solution it can act as a spacer orconnector between protein domains. This may explain the larger shift inBREF51 mobility in the presence of GAD11 protein extract, since GAD11proteins may be forming homodimers connected by the coiled-coil region.GAD11 has a homolog in A. nidulans (E value 6e⁻²⁹), but no homologs inN. crassa were found.

Reverse Transcription PCR (RT-PCR) experiments revealed that while GAD1is expressed constitutively, GAD11 is slightly downregulated in thepresence of 8 mM benzoic acid for 5 h (FIG. 14). While not necessary tounderstand or practice the present invention, if in fact either GAD1 orGAD11 are involved in regulation of the bphA promoter, constitutivetranscription of GAD1 does not rule out the possibility that theactivity of the GAD1 protein is modulated by benzoic acid. On the otherhand, downregulation of the GAD11 gene in the presence of benzoic acidindicates that the protein may function as a transcriptional repressorof the bphA promoter. GAD1 is constitutively expressed, while GAD11seems to be downregulated in the presence of benzoic acid in the media.While not necessary to understand or practice the present invention,there is a possibility that protein-protein interactions play a role inthe regulation of this gene in A. niger.

In some embodiments, plants are transformed with vectors containingconstitutively expressed TFs and maintained as a line to be used inapplications where a novel gene, such as an insect- orpathogen-resistance gene, is to be placed under chemical control. Theselines are then transformed with the gene of interest placed undercontrol of a chimeric promoter. Typically, the TF-containing line isprepared using a hygromycin-selectable marker, whereas the promoter-geneconstruct is prepared using a kanamycin-selectable marker, or viceversa. In other embodiments, plants bearing mutations of a gene ofinterest are transformed with the TF-vectors, which are subsequentlytransformed with the promoter-healthy gene construct, using theindependently selectable markers.

2. External Induction by Benzoate

While not necessary to understand to practice the invention, a morecomplicated mechanism of induction may include a surface receptor thatbinds to benzoate and which then initiates either some kind of signaltransduction cascade or the release of a TF that then culminates in theactivation of the BPH promoter (FIGS. 12C and D). The hypothesis of asurface receptor was tested by using the p-NH₂-benzoate coupled toagarose (as described above) as a non-cleavable external probe. Fungalprotoplasts containing the BRE fused to GFP were then prepared, andactivation of the BRE (production of the GFP marker) by addition of theagarose-immobilized benzoate derivative to the fungal protoplasts wasscored. Whereas the benzoate or p-aminobenzoate induced GFP fluorescencein the BRE-GFP transformed line, no fluorescence was observed whenp-nitrobenzoate was linked to the agarose beads. This initial set ofresults indicates that the TF is a soluble factor. In order toinvestigate whether the disparity of size between the larger agarosebeads and fungal protoplasts may have caused steric problems in bindingof the inducer, smaller ligands still incapable of uptake areconstructed and utilized to confirm a non-membrane surface location.

C. Construction of the Chemically Inducible Hybrid Promoters andPromoter Systems

Following the discovery of the fungal benzoate induced promoter system,which comprises the BRE and transcription factors, an efficientinducible system to control gene expression in cells is engineered; thissystem consists of a gene minimal promoter containing several copies ofthe BREs, and the TFs that bind to these promoter elements.

A chemically inducible promoter is, in some embodiments, constructed byplacing single or multiple copies of a BRE (e.g. BREF51, or a fragmentof SEQ ID NO:6 that is at least 20 nucleotides in length and containsBRE6 TAGTCA (SEQ ID NO:5)), or functionally effective fragments ormodifications thereof, upstream of a minimal gene promoter containing aTATA box transcription start signal, to make the benzoate responseelement functional in cells; the product is referred to as a benzoateinducible hybrid promoter. In some embodiments, additional gene promoterelements may be place around the BREF51, or functionally effectivefragments or modifications thereof, to achieve a host cell-specifictemporal or spatial expression in response to the chemical application.Thus, the present invention provides a benzoate inducible hybridpromoter comprising benzoate-responsive elements within a generegulatory region.

The benzoate inducible hybrid promoter is fused to a reporter gene, suchas GUS (Jefferson et al. (1987) EMBO J. 6: 3901-3907) or GFP (Haseloffet al. (1997) Proc. Natl. Acad. Sci. USA 94: 2122-2127), and transformedinto a cell. The transgenic cells obtained are assayed for theinducibility by expression of the promoter-reporter gene constructalone. It is contemplated that these transgenic cells will not beresponsive to benzoate, and instead provide control levels as the levelof the “leakiness” of the promoter. Northern blots are carried out toverify both the background and the induced levels of expression of thereporter gene, under different concentrations of the inducer.

As determined by the mechanism of induction, at least one additionalplasmid containing at least one gene encoding a transcription factorunder constitutive control is introduced into the transgenic cell (e.g.expressing GAD1 [SEQ ID NO:15, or a fragment thereof determined to alsoserve as a transcription factor] or GAD11 [SEQ ID NO:17, or a fragmentthereof determined to also serve as a transcription factor] or a GAD1homolog (SEQ. ID NO:34), or a GAD11 homolog (SEQ ID NO:36), orfunctional fragments thereof). A constitutive promoter for thetranscription factor ranges from the strong expression of multiple 35Spromoters to lesser levels of expression by plant ubiquitin or actinpromoters. In a simple system, the coding sequence for an entiretranscription factor is operably linked to the promoter, such thatnuclear targeting sequences are present; in more complex systems, codingsequences for enhancers, such as the VP16 and nuclear-targetingsequences (Aoyama and Chua (1997) Plant J. 11: 605-612) are linked tocoding sequences for a truncated version of transcription factors whichcomprises effector and DNA-binding domains of the transcription factor,resulting in a fusion protein factor. The combination of thebenzoate-inducible hybrid promoter and at least one additional geneencoding a transcription factor (which includes but is not limited totranscription factor fusion proteins and other modifications) isreferred to as a benzoate-inducible promoter system. In someembodiments, the transcription factor is GAD1 or GAD11, or functionalfragments thereof (functional fragments can be found using the methodsdescribed above and the Examples using test fragments in place of fulllength GAD1 or GAD11).

The effects, if any, of the inducible system on the overall performanceof the cells are also evaluated.

In some embodiments, the cell to be transformed is a plant cell; infurther embodiments, a transgenic plant cell is regenerated into atransgenic plant. In other embodiments, an Arabidopsis plant istransformed by Agrobacterium-mediated transformation, as described byClough and Bent ((1998) Plant J. 16: 735-743). The transgenic plantsobtained are assayed for the inducibility by expression of thepromoter-reporter gene construct alone. As described above, it iscontemplated that these transgenic plants will not be responsive tobenzoate, and instead provide control levels as the level of the“leakiness” of the promoter. Northern blots are carried out to verifyboth the background and the induced levels of expression of the reportergene, under different concentrations of the inducer.

As determined by the mechanism of induction, at least one additionalplasmid comprising at least one gene encoding a transcription factorunder constitutive control, as described above, is introduced into theseplants.

The effects, if any, of the inducible system on the overall performanceof the plants are also evaluated. Characteristics assessed include butare not limited to pathogen or insect resistance, whole plant and tissueorganization, flowering time, fertility (pollen or egg) or anyphenotypic signs of toxicity. Thus, for example, it is contemplated thatthe inducible system might be utilized to induce male sterility or toovercome it, depending on the application. This could be a very usefulapplication in breeding.

II. Utilization of Hybrid Benzoate Inducible Promoters to ControlExpression of Nucleic Acid Sequences of Interest

The present invention further comprises methods of controllingexpression of nucleic acid sequences of interest using a benzoateinducible hybrid promoter or a benzoate inducible promoter system of thepresent invention. In some embodiments, nucleic acid sequences arecontrolled by a benzoate inducible hybrid promoter of the presentinvention. In other embodiments, nucleic acid sequences are controlledby the benzoate inducible promoter system of the present invention. Thefollowing description is directed to nucleic acid sequences undercontrol of either.

A. Nucleic Acid Sequences of Interest

In some embodiments, the compositions and methods of the presentinvention are used to control or direct nucleic acid sequence expressionin plant seed tissue. Although such sequences are referred to as “genes”under this section, it is understood that these sequences refer to thecoding section of the gene which is expressed as an RNA product, butthat these sequences do not necessarily include the promoter region,although other regulatory regions may be included. In certainembodiments the endogenous promoter region is not included; in others,it may be. The methods are not limited to the control of any particulargene. Indeed, a variety of genes are contemplated for control,including, but not limited to those, described below.

In some embodiments, the gene of interest is an endogenous plant gene.In other embodiments, the gene of interest is an exogenous plant gene.The methods of the present invention are not limited to any particularplant. Indeed, a variety of plants are contemplated, including, but notlimited to angiosperms, gymnosperms, monocotyledons, and dicotyledons.Specific plants contemplated include, but are not limited to, wheat,barley, maize, rye, rice, soybean, hemp, triticale, apricots, oranges,quince, melon, plum, cherry, peach, nectarine, strawberry, grape,raspberry, blackberry, pineapple, papaya, mango, banana, grapefruits,apples, pears, avocados, walnuts, almonds, filberts, pecans, carrots,lettuce, zucchini, tomatoes, beans, peas, cabbage, chicory, onion,garlic, pepper, squash, pumpkin, celery, turnips, radish, spinach,cauliflower, potatoes, sweet potatoes, broccoli, eggplant, cucumber,asparagus, poplar, pine, sequoia, cedar, oak, tobacco, clover, lotus,jojoba, rapeseed, sunflower, sorghum, sugarcane, sugar beet, safflower,Arabidopsis, alfalfa, and cotton. Additional plants include but are notlimited to oats (which are important cereal crop for production ofcholesterol-lowering b-glucans, cassava (which is fifth in world foodcrop production, after corn, wheat, rice, and potatoes), and eucalyptus(an engineered tree species for the pulp and paper industry). Additionalplants include turf grasses, but are not limited to ryegrass, timothy,Bermuda, Bahia, Kentucky bluegrass, zoysia, and the like. Turf grassesare multibillion dollar industries that could benefit from the presentinvention.

In some embodiments, the compositions and methods of the presentinvention are used to control or direct the expression of a geneinvolved in a metabolic pathway of a plant cell (for example, genesresponsible for the synthesis or metabolism of peptides, proteins, fattyacids, lipids, waxes, oils, starches, sugars, carbohydrates, flavors,odors, fragrances, toxins, carotenoid pigments, hormones, cell wallpolymers, gene regulatory molecules, flavonoids, storage proteins,phenolic acids, coumarins, alkaloids, quinones, lignins, glucosinolates,tannins, aliphatic amines, celluloses, pectins, polysaccharides,glycoproteins and glycolipids), in resistance or susceptibility of aplant to diseases (for example, to viral infection), in a visiblephenotype (for example, flower color intensity, color hue and colorpattern); or cell differentiation. For example, specific genescontemplated include, but are not limited to, those described in U.S.Pat. Nos. 5,107,065; 5,283,184; and 5,034,323; each of which is hereinincorporated by reference.

In other embodiments, the compositions and methods of the presentinvention are used to alter the expression of a plant gene whosefunction is unknown in order to elucidate its function. Sense andantisense fragments of the gene are introduced to the plant. The plantis then examined for phenotypic changes (for example, metabolic orvisible).

B. Methods of Transforming Plants

1. Vectors

Nucleic acid sequences of interest intended for expression in plants arefirst assembled in expression cassettes comprising a promoter (forexample, the benzoate inducible hybrid promoter regions of the presentinvention). Methods which are well known to those skilled in the art maybe used to construct expression vectors containing nucleic acidsequences of interest and appropriate transcriptional and translationalcontrol elements. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Such techniques are widely described in the art (See for example,Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., both of which are herein incorporated by reference).

The expression cassettes may further comprise any sequences required forexpression of mRNA. Such sequences include, but are not limited totranscription terminators, enhancers such as introns, viral sequences,and sequences intended for the targeting of the gene product to specificorganelles and cell compartments.

A variety of transcriptional terminators are available for use inexpression of sequences using the promoters of the present invention.Transcriptional terminators are responsible for the termination oftranscription beyond the transcript and its correct polyadenylation.Appropriate transcriptional terminators and those which are known tofunction in plants include, but are not limited to, the CaMV 35Sterminator, the tml terminator, the pea rbcS E9 terminator, and thenopaline and octopine synthase terminator (See for example, Odell etal., Nature 313:810 (1985); Rosenberg et al., Gene, 56:125 (1987);Guerineau et al., Mol. Gen. Genet., 262:141 (1991); Proudfoot, Cell,64:671 (1991); Sanfacon et al., Genes Dev., 5:141; Mogen et al., PlantCell, 2:1261 (1990); Munroe et al., Gene, 91:151 (1990); Ballas et al.,Nucleic Acids Res. 17:7891 (1989); Joshi et al., Nucleic Acid Res.,15:9627 (1987)).

In addition, in some embodiments, constructs for expression of a nucleicacid sequence of interest include one or more of sequences found toenhance gene expression from within the transcriptional unit. Thesesequences can be used in conjunction with the nucleic acid sequence ofinterest to increase expression in plants. Various intron sequences havebeen shown to enhance expression, particularly in monocotyledonouscells. For example, the introns of the maize Adh1 gene have been foundto significantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells (Callis et al., GenesDevelop. 1: 1183 (1987)). Intron sequences have been routinelyincorporated into plant transformation vectors, typically within thenon-translated leader.

In some embodiments of the present invention, the construct forexpression of the nucleic acid sequence of interest also includes aregulator such as a nuclear localization signal (Kalderon et al., Cell39:499 (1984); Lassner et al., Plant Molecular Biology 17:229 (1991)), aplant translational consensus sequence (Joshi, Nucleic Acids Research15:6643 (1987)), an intron (Luehrsen and Walbot, Mol. Gen. Genet. 225:81(1991)), and the like, operably linked to the nucleic acid sequence ofinterest.

In preparing the construct comprising the nucleic acid sequence ofinterest, various DNA fragments can be manipulated, so as to provide forthe DNA sequences in the desired orientation (for example, sense orantisense) orientation and, as appropriate, in the desired readingframe. For example, adapters or linkers can be employed to join the DNAfragments or other manipulations can be used to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resection, ligation, or the like ispreferably employed, where insertions, deletions or substitutions (forexample, transitions and transversions) are involved.

Numerous transformation vectors are available for plant transformation.The selection of a vector for use will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers are preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing and Vierra, Gene 19: 259(1982); Bevan et al., Nature 304:184 (1983)), the bar gene which confersresistance to the herbicide phosphinothricin (White et al., Nucl AcidsRes. 18:1062 (1990); Spencer et al., Theor. Appl. Genet. 79: 625(1990)), the hph gene which confers resistance to the antibiotichygromycin (Blochlinger and Diggelmann, Mol. Cell. Biol. 4:2929 (1984)),and the dhfr gene, which confers resistance to methotrexate (Bourouis etal., EMBO J., 2:1099 (1983)).

In some embodiments of the present invention, transformation is carriedout using Agrobacterium tumefaciens mediated methods. Many vectors areavailable for transformation using Agrobacterium tumefaciens. Thesetypically carry at least one T-DNA border sequence and include vectorssuch as pBIN19 (Bevan, Nucl. Acids Res., 12:8711 (1984)). An additionalvector useful for Agrobacterium-mediated transformation is the binaryvector pCIB10 (Rothstein et al., Gene 53:153 (1987)) which contains agene encoding kanamycin resistance for selection in plants, T-DNA rightand left border sequences and incorporates sequences from the widehost-range plasmid pRK252 allowing it to replicate in both E. coli andAgrobacterium. Various derivatives of pCIB10 have been constructed whichincorporate the gene for hygromycin B phosphotransferase (See forexample, Gritz et al., Gene, 25: 179 (1983)). These derivatives enableselection of transgenic plant cells on hygromycin only (pCIB743), orhygromycin and kanamycin (pCIB715, pCIB717).

In some embodiments of the present invention, the nucleic acid sequenceof interest is introduced directly into a plant. One vector useful fordirect gene transfer techniques in combination with selection by theherbicide Basta (or phosphinothricin) is a modified version of theplasmid pCIB246, with the CaMV 35S promoter replaced by a benzoateinducible hybrid promoter of the present invention (as described above)in operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in WO 93/07278, which isherein incorporated by reference. In some embodiments of the presentinvention, this vector is modified to include a benzoate induciblehybrid promoter of the present invention (as described above)operatively linked to two nucleic acid sequences of interest. The geneproviding resistance to phosphinothricin is the bar gene fromStreptomyces hygroscopicus (Thompson et al., EMBO J., 6:2519 (1987)).

2. Transformation Techniques

Once the nucleic acid sequences have been operatively linked to abenzoate inducible hybrid promoter of the present invention and insertedinto a suitable vector for the particular transformation techniqueutilized (for example, one of the vectors described above), therecombinant DNA described above can be introduced into a plant cell in anumber of art-recognized ways. Those skilled in the art will appreciatethat the choice of method depends upon the type of plant targeted fortransformation. In some embodiments, the vector is maintainedepisomally. In other embodiments, the vector is integrated into thegenome. In some embodiments, plants are independently transformed into aline carrying the constitutively expressed transcription factor (ormodification or fusion thereof), or the benzoate inducible hybridpromoter-target gene construct is transformed into a line of choice andcrossed with lines carrying the transcription factor (or modification orfusion thereof), back-crossed and selected for progeny carrying bothconstructs.

In some embodiments, vectors useful in the practice of the presentinvention are microinjected directly into plant cells by use ofmicropipettes to mechanically transfer the recombinant DNA (Crossway,Mol. Gen. Genet, 202:179 (1985)). In still other embodiments, the vectoris transferred into the plant cell by using polyethylene glycol (Krenset al., Nature, 296:72 (1982); Crossway et al., BioTechniques, 4:320(1986)); fusion of protoplasts with other entities, either minicells,cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al.,Proc. Natl. Acad. Sci., USA, 79:1859 (1982)); protoplast, transformation(EP 0 292 435; herein incorporated by reference); direct gene transfer(Paszkowski et al., EMBO J., 3:2717 (1984); Hayashimoto et al., PlantPhysiol. 93:857 (1990)).

In other embodiments, the vector may also be introduced into the plantcells by electroporation. (Fromm, et al., Pro. Natl Acad. Sci. USA82:5824, 1985; Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602 (1986)).In this technique, plant protoplasts are electroporated in the presenceof plasmids containing the gene construct. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form plant callus.

In other embodiments, DNA is introduced into plant cells by what iscalled the “pollen-tube pathway’ whereby naked DNA plasmids orAgrobacterium-mediated transfer is made at the time of anthesis bydipping the flowers into a solution of the plasmid-containing bacteriaor by spraying the plasmid-containing bacteria as an aerosol onto theflowers (Clough and Bent (1998) Plant J. 16: 735-743; Bent (2000) PlantPhysiol. 124: 1540-1547). In other embodiments, plants are transformedby cutting styles on the morning of anthesis and droplets of solutioncontaining the DNA are added directly to the cut ends (Huang et al.(1998) Chin. Sci. Bull. 44: 698-708; Zeng et al. (1998) Chin. Sci. Bull.43: 798-803; Hu and Wang (1999) In Vitro Cell. Devel. Biol.-Plant 35:417-420; Tjokrokusumo et al. (2000) Plant Cell Rep. 19: 792-797; andHerrero (2001) Sex. Plant Reprod. 14: 3-7). In each of theseembodiments, the pollen or egg cells are transformed coincident withfertilization, and the successful transformants selected upongermination.

In still further embodiments, the vector is introduced through ballisticparticle acceleration using devices (for example, available fromAgracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.). (Seefor example, U.S. Pat. No. 4,945,050; herein incorporated by reference;and McCabe et al., Biotechnology 6:923 (1988)). See also, Weissinger etal., Annual Rev. Genet. 22:421 (1988); Sanford et al., ParticulateScience and Technology, 5:27 (1987) (onion); Svab et al., Proc. Natl.Acad. Sci. USA, 87:8526 (1990) (tobacco chloroplast); Christou et al.,Plant Physiol., 87:671 (1988) (soybean); McCabe et al., Bio/Technology6:923 (1988) (soybean); Klein et al., Proc. Natl. Acad. Sci. USA,85:4305 (1988) (maize); Klein et al., Bio/Technology, 6:559 (1988)(maize); Klein et al., Plant Physiol., 91:4404 (1988) (maize); Fromm etal., Bio/Technology, 8:833 (1990); and Gordon-Kamm et al., Plant Cell,2:603 (1990) (maize); Koziel et al., Biotechnology, 11:194 (1993)(maize); Hill et al., Euphytica, 85:119 (1995) and Koziel et al., Annalsof the New York Academy of Sciences 792:164 (1996); Shimamoto et al.,Nature 338: 274 (1989) (rice); Christou et al., Biotechnology, 9:957(1991) (rice); Datta et al., Bio/Technology 8:736 (1990) (rice);European Patent Application EP 0 332 581, herein incorporated byreference (orchard grass and other Pooideae); Vasil et al.,Biotechnology, 11: 1553 (1993) (wheat); Weeks et al., Plant Physiol.,102: 1077 (1993) (wheat); Wan et al., Plant Physiol. 104: 37 (1994)(barley); Knudsen and Muller, Planta, 185:330 (1991) (barley); Umbeck etal., Bio/Technology 5: 263 (1987) (cotton); Casas et al., Proc. Natl.Acad. Sci. USA 90:11212 (1993) (sorghum); Somers et al., Bio/Technology10:1589 (1992) (oat); Torbert et al., Plant Cell Reports, 14:635 (1995)(oat); Weeks et al., Plant Physiol., 102:1077 (1993) (wheat); and Changet al., WO 94/13822 (wheat).

In addition to direct transformation, in some embodiments, the vectorscomprising the nucleic acid sequences of interest and a promoter of thepresent invention are transferred using Agrobacterium-mediatedtransformation (Hinchee et al., Biotechnology, 6:915 (1988); Ishida etal., Nature Biotechnology 14:745 (1996)). Agrobacterium is arepresentative genus of the gram-negative family Rhizobiaceae. Itsspecies are responsible for plant tumors such as crown gall and hairyroot disease. In the dedifferentiated tissue characteristic of thetumors, amino acid derivatives known as opines are produced andcatabolized. The bacterial genes responsible for expression of opinesare a convenient source of control elements for chimeric expressioncassettes. Heterologous genetic sequences (for example, nucleic acidsequences operatively linked to a promoter of the present invention),can be introduced into appropriate plant cells, by means of the Tiplasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted toplant cells on infection by Agrobacterium tumefaciens, and is stablyintegrated into the plant genome (Schell, Science, 237: 1176 (1987)).Species which are susceptible infection by Agrobacterium may betransformed in vitro.

3. Regeneration

After determination of the presence and expression of the desired geneproducts, whole plants are regenerated. Plant regeneration from culturedprotoplasts is described in Evans et al., Handbook of Plant CellCultures, Vol. 1: (MacMillan Publishing Co. New York, 1983); and VasilI. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad.Press, Orlando, Vol. I, 1984, and Vol. III, 1986. It is known that manyplants can be regenerated from cultured cells or tissues, including bothmonocots and dicots, and including for example, crop plants, ornamentalsand other horticultural plants, shrubs, and trees. Means forregeneration vary from species to species of plants, but generally asuspension of transformed protoplasts is first provided. Callus tissueis formed and shoots may be induced from callus and subsequently rooted.

Alternatively, embryo formation can be induced from the protoplastsuspension. These embryos germinate and form mature plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. Shoots and roots normally develop simultaneously.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. The reproducibility of regenerationdepends on the control of these variables.

C. Increasing or Decreasing Gene Expression

It is contemplated that benzoate inducible hybrid promoters and/orbenzoate inducible promoter systems of the present invention may beutilized to either increase or decrease the level of expression ofnucleic acid sequences of interest in transfected cells as compared tothe levels in wild-type cells. Accordingly, in some embodiments,expression in plants by the methods described above leads to theoverexpression of nucleic acid sequences of interest in transgenicplants, plant tissues, or plant cells.

In other embodiments of the present invention, benzoate inducible hybridpromoters and/or benzoate inducible promoter systems of the presentinvention are utilized to decrease the level of expression of nucleicacid sequences of interest in transgenic plants, plant tissues, or plantcells as compared to wild-type plants, plant tissues, or plant cells.One method of reducing expression utilizes expression of antisensetranscripts. Antisense RNA has been used to inhibit plant target genesin a tissue-specific manner (for example, van der Krol et al.,Biotechniques 6:958-976 (1988)). Antisense inhibition has been shownusing the entire cDNA sequence as well as a partial cDNA sequence (forexample, Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988);Cannon et al., Plant Mol. Biol. 15:39-47 (1990)). There is also evidencethat 3′ non-coding sequence fragment and 5′ coding sequence fragments,containing as few as 41 base-pairs of a 1.87 kb cDNA, can play importantroles in antisense inhibition (Ch'ng et al., Proc. Natl. Acad. Sci. USA86:10006-10010 (1989)).

Accordingly, in some embodiments, benzoate inducible hybrid promoters ofthe present invention (see, for example, FIG. 15) are operably linked tonucleic acid sequences of interest which are oriented in a vector andexpressed so as to produce antisense transcripts. To accomplish this, anucleic acid segment from the desired gene is cloned and operably linkedto a benzoate inducible hybrid promoter of the present invention suchthat the antisense strand of RNA will be transcribed. The expressioncassette is then transformed into plants, by itself or as part of thebenzoate inducible hybrid promoter system, and the antisense strand ofRNA is produced in response to application of the chemical inducer. Thenucleic acid segment to be introduced generally will be substantiallyidentical to at least a portion of the endogenous gene or genes to berepressed. The sequence, however, need not be perfectly identical toinhibit expression. The vectors of the present invention can be designedsuch that the inhibitory effect applies to other proteins within afamily of genes exhibiting homology or substantial homology to thetarget gene.

Furthermore, for antisense suppression, the introduced sequence alsoneed not be full length relative to either the primary transcriptionproduct or fully processed mRNA. Generally, higher homology can be usedto compensate for the use of a shorter sequence. Furthermore, theintroduced sequence need not have the same intron or exon pattern, andhomology of non-coding segments may be equally effective. Normally, asequence of between about 30 or 40 nucleotides and about full lengthnucleotides should be used, though a sequence of at least about 100nucleotides is preferred, a sequence of at least about 200 nucleotidesis more preferred, and a sequence of at least about 500 nucleotides isespecially preferred.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of the target gene or genes. It is possible to designribozymes that specifically pair with virtually any target RNA andcleave the phosphodiester backbone at a specific location, therebyfunctionally inactivating the target RNA. In carrying out this cleavage,the ribozyme is not itself altered, and is thus capable of recycling andcleaving other molecules, making it a true enzyme. The inclusion ofribozyme sequences within antisense RNAs confers RNA-cleaving activityupon them, thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs which arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, Solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff, et al., Nature 334:585-591 (1988).

Another method of reducing expression of nucleic acid sequences ofinterest utilizes the phenomenon of cosuppression or gene silencing (Seefor example, U.S. Pat. No. 6,063,947, incorporated herein by reference).The phenomenon of cosuppression has also been used to inhibit planttarget genes in a tissue-specific manner. Cosuppression of an endogenousgene using a full-length cDNA sequence as well as a partial cDNAsequence (730 bp of a 1770 bp cDNA) are known (for example, Napoli etal., Plant Cell 2:279-289 (1990); van der Krol et al., Plant Cell2:291-299 (1990); Smith et al., Mol. Gen. Genetics 224:477-481 (1990)).Accordingly, in some embodiments the benzoate inducible hybrid promoterof the present invention are operably linked to nucleic acid sequencesof interest which are expressed in another species of plant, eitheralone or as part of the benzoate inducible hybrid system, to effectcosuppression of a homologous gene.

Generally, where inhibition of expression is desired, some transcriptionof the introduced sequence occurs. The effect may occur where theintroduced sequence contains no coding sequence per se, but only intronor untranslated sequences homologous to sequences present in the primarytranscript of the endogenous sequence. The introduced sequence generallywill be substantially identical to the endogenous sequence intended tobe repressed. This minimal identity will typically be greater than about65%, but a higher identity might exert a more effective repression ofexpression of the endogenous sequences. Substantially greater identityof more than about 80% is preferred, though about 95% to absoluteidentity would be most preferred. As with antisense regulation, theeffect should apply to any other proteins within a similar family ofgenes exhibiting homology or substantial homology.

For cosuppression, the introduced sequence in the expression cassette,needing less than absolute identity, also need not be full length,relative to either the primary transcription product or fully processedmRNA. This may be preferred to avoid concurrent production of someplants which are overexpressers. A higher identity in a shorter thanfull length sequence compensates for a longer, less identical sequence.Furthermore, the introduced sequence need not have the same intron orexon pattern, and identity of non-coding segments will be equallyeffective. Normally, a sequence of the size ranges noted above forantisense regulation is used.

In some embodiments, the nucleic acid sequence of interest is an siRNA(RNAi) molecule that is able to suppress a targeted RNA transcript. RNAirepresents an evolutionary conserved cellular defense for controllingthe expression of foreign genes in most eukaryotes, including humans.RNAi is triggered by double-stranded RNA (dsRNA) and causessequence-specific mRNA degradation of single-stranded target RNAshomologous in response to dsRNA. The mediators of mRNA degradation aresmall interfering RNA duplexes (siRNAs), which are normally producedfrom long dsRNA by enzymatic cleavage in the cell. siRNAs are generallyapproximately twenty-one nucleotides in length (e.g. 21-23 nucleotidesin length), and have a base-paired stricture characterized by twonucleotide 3′-overhangs. Following the introduction of a small RNA, orRNAi, into the cell, it is believed the sequence is delivered to anenzyme complex called RISC (RNA-induced silencing complex). RISCrecognizes the target and cleaves it with an endonuclease. It is notedthat if larger RNA sequences are delivered to a cell, RNase III enzyme(Dicer) converts longer dsRNA into 21-23 nt ds siRNA fragments.

Chemically synthesized siRNAs have become powerful reagents forgenome-wide analysis of mammalian gene function in cultured somaticcells. Beyond their value for validation of gene function, siRNAs alsohold great potential as gene-specific therapeutic agents (Tuschl andBorkhardt, Molecular Intervent. 2002; 2(3):158-67, herein incorporatedby reference).

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:4948; Elbashir et al., Genes Dev. 2001; 15: 188-200; andElbashir et al., EMBO J. 2001; 20:6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are described, for example, in U.S. Pat. No. 6,506,559,herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference.

In other embodiments, the nucleic acid sequence of interest is a geneconversion gene (e.g. to direct the change in an allele in a cell).Methods for designing and employing gene conversion genes are describedin U.S. Pat. Pub. US20030051270 (herein incorporated by reference).

III. Methods of Production of Gene Products of Interest

The present invention further comprises methods of producing products ofnucleic acid sequences of interest by using benzoate inducible hybridpromoters and/or benzoate inducible hybrid promoter systems of thepresent invention. Products of nucleic acid sequences include proteins,RNA, and metabolic products or catalytically active proteins or RNAs,such as secondary metabolites, sugars, lipids, modified proteins, andnucleic acids.

A. Production in Plants

In some embodiments, the present invention provides methods of producingone or more gene products of interest using a benzoate inducible hybridpromoter and/or benzoate inducible hybrid promoter system of the presentinvention. In some embodiments, a benzoate inducible hybrid promoter ofthe present invention (as described above) is used to express two geneproducts of interest (for example, two subunits of a multi-subunitprotein or two members of a metabolic pathway) from the same promoterconstruct. In other embodiments, a sequence that hybridizes to abenzoate inducible hybrid promoter of the present invention is utilized.One skilled in the art will recognize, in view of the presentdisclosure, that the expression vectors comprising a benzoate induciblehybrid promoter of the present invention and one or more nucleic acidsequences of interest may contain additional regulatory and enhancerelements specific to the host cell utilized for expression (for example,those described above or below).

In some embodiments, one or more gene products of interest are expressedin regenerated plants (for example, in seed tissue to elicit a specificmetabolic response). In other embodiments, polypeptides of interest areexpressed in plants for use in food stuffs (for example, to increase thenutritional value or to express a pharmaceutical compound). In stillfurther embodiments, one or more polypeptides of interest are expressedin cell culture (for example, plant, bacterial, or eukaryotic cells) forthe purpose of purifying the polypeptides of interest from the cellculture.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elementsmay be utilized. For example, for expression mediated by plant viruses,viral promoters or leader sequences may be included in the vector.

In some preferred embodiments, the 5′ leader sequence is included in theexpression cassette construct. Such leader sequences can act to enhancetranslation. Translation leaders are known in the art and include:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′non-coding region; Elroy-Stein et al., PNAS, 86:6126 (1989)); potyvirusleaders, for example, TEV leader (Tobacco Etch Virus; Niepel and Gallie,J Virol., 73:9080 (1999)) MDMV leader (Maize Dwarf Mosaic Virus;Virology, 154:9 (1986)), and human immunoglobulin heavy-chain bindingprotein (BiP; Macejak and Samow, Nature 353:90 (1991)); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4;Jobling and Gebrke, Nature, 325:622 (1987)); tobacco mosaic virus leader(TMV; Gallie et al., Molecular-Biology of RNA, pages 237-256 (1989));and maize chlorotic mottle virus leader (MCMV; Lommel et al., Virology91:382 (1991); Della-Cioppa et al., Plant Physiology 84:965 (1987)).

In some embodiments, one or more polypeptides of interest are expressedin plants using stable transformation, as described above. In otherembodiments, plant vectors are created using a recombinant plant viruscontaining a recombinant plant viral nucleic acid, as described in PCTpublication WO 96/40867 which is herein incorporated by reference.Subsequently, the recombinant plant viral nucleic acid which containsone or more nucleic acid sequences encoding polypeptides of interest aretranscribed or expressed in the infected tissues of the plant host andthe polypeptides are recovered from the plant, as described in WO99/36516, which is herein incorporated by reference.

In this embodiment, recombinant plant viral nucleic acids which containa benzoate inducible hybrid promoter of the present invention linked toat least one nucleic acid sequence of interest are utilized. Therecombinant plant viral nucleic acids have substantial sequence homologyto plant viral nucleic acid sequences and may be derived from an RNA,DNA, cDNA or a chemically synthesized RNA or DNA. A partial listing ofsuitable viruses is described below.

The first step in producing recombinant plant viral nucleic acidsaccording to this particular embodiment is to modify the nucleic acidsequences of the plant viral nucleic acid sequence by known techniquessuch that a benzoate inducible hybrid promoter of the present inventionis inserted into the plant viral nucleic acid without destroying thebiological function of the plant viral nucleic acid. The native coatprotein coding sequence may be deleted in some embodiments, placed underthe control of a non-native subgenomic promoter in other embodiments, orretained in a further embodiment. If it is deleted or otherwiseinactivated, a non-native coat protein gene is inserted under control ofone of the non-native subgenomic promoters, or optionally under controlof the native coat protein gene subgenomic promoter. The non-native coatprotein is capable of encapsidating the recombinant plant viral nucleicacid to produce a recombinant plant virus. Thus, the recombinant plantviral nucleic acid contains a coat protein coding sequence, which may benative or a nonnative coat protein coding sequence, under control of oneof the native or non-native subgenomic promoters. The coat protein isinvolved in the systemic infection of the plant host.

Some of the viruses suitable for use in the present invention include,but are not limited to viruses from the tobamovirus group such asTobacco Mosaic virus (TMV), Ribgrass Mosaic Virus (RGM), Cowpea Mosaicvirus (CMV), Alfalfa Mosaic virus (AMV), Cucumber Green Mottle Mosaicvirus watermelon strain (CGMMV-W) and Oat Mosaic virus (OMV) and virusesfrom the brome mosaic virus group such as Brome Mosaic virus (BMV),broad bean mottle virus and cowpea chlorotic mottle virus. Additionalsuitable viruses include Rice Necrosis virus (RNV), and geminivirusessuch as tomato golden mosaic virus (TGMV), Cassava latent virus (CLV)and maize streak virus (MSV).

Other embodiments of plant vectors used for the expression of sequencesencoding polypeptides include, for example, a benzoate inducible hybridpromoter of the present invention used in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307 (1987)). Theseconstructs can be introduced into plant cells by any suitable methods,including, but not limited to those described above.

B. Confirmation of Product Presence

Host cells which contain a nucleic acid sequence of interest may beidentified by a variety of procedures known to those of skill in theart. These procedures include, but are not limited to, enzyme assay,DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassaytechniques which include membrane, solution, or chip based technologiesfor the detection and/or quantitation of nucleic acid or protein.

The presence of nucleic acid sequences of interest can be detected byDNA-DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding the polypeptide.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences of interest todetect transformants containing DNA or RNA encoding the polypeptide.

A variety of protocols for detecting and measuring the expression of apolypeptide using either polyclonal or monoclonal antibodies specificfor the protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson the polypeptide is preferred, but a competitive binding assay may beemployed. These and other assays are described, among other places, inHampton et al. (1990); Serological Methods, a Laboratory Manual, APSPress, St Paul, Minn.; and Maddox et al., J. Exp. Med., 158:1211(1983)).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding a polypeptide ofinterest include oligonucleotide labeling, nick translation,end-labeling or PCR amplification using a labeled nucleotide.Alternatively, the sequences encoding the polypeptide, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits from Pharmacia & Upjohn (Kalamazoo, Mich.), Promega Corporation(Madison, Wis.) and U.S. Biochemical Corp. (Cleveland, Ohio). Suitablereporter molecules or labels, which may be used, includeradionucleotides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

C. Recovery of Expressed Products

In some embodiments of the present invention, it is desirable to recoverexpressed protein from seed tissue. In other embodiments, it isdesirable to recover expressed protein from floral, leaf, stem, root, orother tissues. Plants transformed with nucleic acid sequences encodingone or more polypeptides of interest may be cultivated under conditionssuitable for high expression and subsequent recovery of the protein fromthe appropriate tissue. The protein produced by a recombinant cell maybe secreted or contained intracellularly depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing polynucleotides which encode thepolypeptide(s) of interest may be designed to contain signal sequenceswhich direct secretion of the polypeptide into a particular cellcompartment, such as a vacuole or a plastid, or secretion from the cellinto the extracellular matrix or cell wall.

In other embodiments of the present invention, other recombinantconstructions may be used to join sequences encoding a polypeptide tonucleic acid sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (available from Invitrogen, San Diego, Calif.)between the purification domain and the polypeptide of interest may beused to facilitate purification. One such expression vector provides forexpression of a fusion protein containing the polypeptide of interestand a nucleic acid encoding 6 histidine residues preceding a thioredoxinor an enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography) asdescribed in Porath et al., Prot. Exp. Purif., 3:263 (1992) while theenterokinase cleavage site provides a means for purifying thepolypeptide from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll et al., DNA Cell Biol.,12:441 (1993)).

In yet other embodiments, it is desirable to recover other products ofinterest, such as RNA, or products of metabolically active proteins,such as secondary metabolites, sugars, lipids, modified proteins, andnucleic acids. Such products are recovered by methods well known in theart.

IV. Chemical Induction of Flowering

The chemically inducible promoter system developed for use in plants, asdescribed above, can be utilized to control a developmental phase inplants. In one aspect of the present invention, a chemically induciblepromoter system developed for use in plants is used to control orregulate flowering in plants. The first step is the development of amodel that employs a chemically inducible promoter and requiredtranscription factors to control flowering in plants.

A. A Test Model for Induction of Flowering by a Chemically InducedPromoter

One of the more practical uses for a chemically inducible promoter is tocontrol a plant developmental process. For many decades, plantphysiologists have been attempting to elucidate the mechanism by whichflowering is controlled. Much of this work has been focused onidentifying the specific plant growth regulators produced in the leavesand transported to the apical and lateral meristems where they induce,singly or in combination, a change from a vegetative to a floraldevelopmental program. The elusive florigen has never been identified.However, a gene has been identified in Arabidopsis that appears to beone of several key transcriptional control factors necessary fortransition of the meristem to flowering mode. The gene, called LEAFY,was identified as a mutation that markedly delayed the onset offlowering (Schultz and Haughn (1991) Plant Cell 3, 771-781; Weigel etal. (1992) Cell 69, 843-859). This transition is controlled by severalgenetic pathways that impact the change in developmental state (Levy andDean 1998). Another such factor is a floral inducer called FLOWERINGLOCUS T (FT), a mutant of which also causes late flowering (Koorneef etal. (1991) Mol. Gen. Genet. 229: 57-66). Activation tagging of FT, whichcauses constitutive expression of the FT, induces precocious floweringat the three-leaf stage of seedling development (Kardailsky et al.(1999) Science 286: 1962-1965). This gene works in parallel with LEAFY,but obviously exerts marked control of the timing of flowering.

The ability of a chemically controllable promoter to induce floweringwas tested in Arabidopsis containing a disabled FT gene. The ft-3 mutantArabidopsis, which is caused by missense mutation deleting the entireC-terminal region of the protein and displays a significant delay in theflowering response, was obtained from the Arabidopsis Stock Center (OhioState Univ).

A chemically inducible promoter available to test induction of floweringis the glucocorticoid receptor-promoter element system that is inducedby the glucocorticoid, dexamethasone (Aoyama and Chua (1997) Plant J.11: 605-612; Kunkel et al. (1999) Nature Biotech. 17: 916-919). Thissystem has recently been engineered to avoid some leakiness problemsoriginally experienced, and the plasmid constructs for the two necessarycomponents have been obtained. The general strategy of theglucocorticoid receptor promoter was described by Aoyama and Chua((1997) Plant J. 11, 605-612). Dr. Jen Sheen (Massachusetts Generalhospital) restructured the glucocorticoid promoter to be LexA-basedinstead of Gal4, and this provided a finer control. This promoter can beassembled from available sequences by one skilled in the art.

Wild-type FT cDNA was obtained from Dr. Detlef Weigel (formerly of theSalk Institute, La Jolla, Calif., and now Max Planck Institute forDevelopmental Biology, Tübingen, Germany); the wild-type FT cDNA wasprepared from transcripts for flowering Arabidopsis plants. This andother Arabidopsis cDNAs are available from Dr. Weigel or from a publiclyaccessible Riken BioResource Center, Japan at the riken web site. As theArabidopsis genome is completely sequenced and publicly available, oneskilled in the art can design primers and obtain a full-length FT geneby RT-PCR. Wild-type FT cDNA was placed under the control of theLexA-based glucocorticoid inducible promoter (called Lex-FT), and thisconstruct was subcloned into the pCAMBIA 1305.1 binary vector (CAMBIA,Canberra, Australia). In a separate construct, the hybridtransactivating factor LSVG, under a constitutively expressed promoter,was cloned into pCAMBIA 1302 (CAMBIA, Canberra, Australia). Both vectorswere then transformed by vacuum infiltration (Clough and Bent (1998)Plant J. 16: 735-743) into the Arabidopsis ft plants. Ti seeds wereharvested, and plated out on selective media, containing the antibiotichygromycin, in order to recover putative transformants.

The selected transformants have been assayed for GUS or GFP expression(depending on the pCAMBIA vector used), and the presence of theinducible promoter system and the FT gene have been confirmed by PCR. Aproblem was encountered with the plants containing the pCAMBIA 1302-LSVGconstitutive-expressant construct, probably due to a slow accumulationof toxic levels the hybrid transacting factor LSVG during the prolongedgrowth before flowering. The transgenic plants died shortly afterflowering, yielding few or no seeds from the plants. To overcome thisunexpected difficulty with this test system, the LSVG hybridtransactivator was subcloned into a different pCAMBIA vector, with akanamycin selectable marker instead of hygromycin. Confirmed Lex-FThomozygous plants are transformed with the new construct. This approachprovides not only a faster way of incorporating both components into thesame plant, but also overcomes the toxicity problem by spraying the testplants after only one-third the growth period.

As described above, the Lex-FT construct has been successfullyintegrated into non-leaky ft homozygous mutants; these plants are thentransformed with the altered construct containing a constitutivelyexpressed LSVG in a binary vector with kanamycin resistance.

B. Induction of Flowering by the Hybrid Promoter System Described Above

The dexamethasone inducible promoter system described above is replacedwith a benzoate-inducible promoter system, as described above.

An embodiment of the benzoate inducible hybrid promoter system is shownin FIG. 15. The first DNA construct is the Inducible Promoter itself,with 3 or more copies of BREF51 (BRE), fused to the TATA-box of theCauliflower Mosaic Virus 35S promoter (35S TATA). A polycloning site isengineered between the 35S TATA promoter sequence and the NopalineSynthase (nos) terminator for ease of manipulation. This hybrid promoterdrives expression of any downstream gene of interest in a benzoateinducible manner. The nos terminator is placed downstream of the codingregion and, in addition to stop codons in every reading frame, containsa polyadenylation signal sequence.

The second DNA construct is the Transacting Factor or TranscriptionalFactor, which will be constitutively expressed by using a full-lengthCaMV35S promoter. A chimeric transcription factor construct may also beemployed that consists of a combination of modules. The first module isthe Nuclear Localization Signal (NLS) from the SV40 viral protein. TheActivation Domain (AD) of the Herpes Simplex Viral Protein 16 (VP16)constitutes the second module. The third and final module have theBREF51 DNA binding domain (BRE DBD) fused to the benzoate receptordomain (Benz Rec), both identified from the A. niger transcriptionfactor. The nos terminator sequence is also be placed downstream of thisconstruct.

V. Exemplary Applications

There is an avid interest to precisely control the timing and level ofexpression of transgenes in plants and other cell types. Such controlhas great utility in both basic research and in agriculture. Thus, thehybrid benzoate inducible promoter system described above has utility inboth research and agricultural applications. The promoter system can beused to control expression of exogenous genes, of endogenous genes, andof antisense genes targeted to either or both exogenous or endogenousgenes.

In research, using mutants to understand unknown gene function isseverely limited when such mutations are lethal. Placing such genesunder inducible control of the hybrid benzoate inducible promoter systemdescribed above constitutes a powerful solution to this problem.Spraying a plant with an innocuous chemical and switching on a gene ofinterest provides a powerful tool for basic studies, such as phenotypesassociated with specific gene expression, as well as gene interactionsin plants.

Moreover, practical and large-scale applications are also anticipatedfor the hybrid benzoate inducible promoter system described above.Turning on engineered plant defense genes only upon attack of thepathogen or insect is anticipated to save millions of dollars inpesticide application; the precise timing flowering in greenhouseoperations is anticipated to not only save money, but to open upadditional market products to the floricultural industries. As benzoateis degraded easily and is non-toxic, it can be used in the field as apotent inducer of agronomic fruits, such as processing tomatoes, whichare collected mechanically in a single harvest. Precision timing offlowering will better synchronize the flower and subsequent fruitproduction so that a single harvest method will result in higher yields.Precise control of transgene expression in a specific plant tissue hasbeen accomplished by means of using several different tissue-specificpromoters. Another contemplated application is the use of the benzoateinducible hybrid promoter in control of fertility, where a pollensterility gene under control of the hybrid promoter could be induced orsilenced, depending on the desire of the breeder.

Hence, the present invention provides the hybrid benzoate induciblepromoter system in a kit for sale by molecular biology supply companies.In some embodiments, a kit comprises two plasmids: a first binaryplasmid with a range of constitutively expressing promoters capable ofcontrolling expression of at least one benzoate-activatabletranscription factor and a hygromycin resistance selectable marker; anda second binary plasmid comprising a benzoate inducible promoter asdescribed above, an efficient polycloning site for insertion of a codingsequence under control of the benzoate inducible promoter, and aterminator coupled with a kanamycin-resistance marker. One skilled inthe art utilizes the components of the kit to carry out thetransformations. In some embodiments, the plasmids are suitablyengineered for a particular host; for example, the plasmids and benzoateinducible promoter are capable of transformation of and expression inplants, as described above.

Such kits are contemplated to find use in both the research lab, and inthe development of agricultural plants where timing of control of geneexpression is desired.

Precise control of gene expression is equally important in animal andfungal systems with respect to understanding the function of genes forwhich mutations would otherwise be lethal. For example, to determine ifmutations that would be lethal at particular stages of organismdevelopment, antisense (or RNAi) constructions of the gene could be keptsilent until induction was desired. Although controlling in vitrodevelopment in cell cultures would be more facile, because benzoate isnon-toxic, its use in vivo in animal model systems is feasible. In otherapplications, the function of genes suspected to be mutated in animaland fungal model systems could be tested by placing a complementary geneunder control by the chemical. The basic benzoate activation systemwould be the same, but other minimal promoters and selective media wouldbe employed that are optimal for non-plant systems.

For all organisms, plant, animal and fungal, further refinements of thebenzoate-inducible promoter system could be made to increase sensitivityto the inducing compound, such as in fusion of the transcription factorwith enhanced nuclear localization signals or amplification ofactivation through simultaneous induction of the transcription factor ina benzoate-specific manner.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosures which follow, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); fmoles (femtomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); l or L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); BPH (benzoatepara-hydroxylase); Benzoate Response Element (BRE); BREF51 (BenzoateResponse Factor containing Promoter fragment of 51 bp); double strandedBRE51 fragment containing a poly(A) overhang (BREaff); transcriptionfactor (TF); Electrophoretic Mobility-Shift Assays (EMSA); GreenFluorescent Protein (GFP); paramagnetic particles (PMPs); Streptavidincoupled to Paramagnetic Particles (SA-PMPs); matrix-assistedlaser-desorption ionization—time of flight (MALDI-TOF MS); Saline SodiumCitrate (SSC.)

Example 1 Materials and Methods

This example describes the materials and methods used to identify thepromoter region of the bphA gene, to identify the benzoate responseelements, and to identify associated factors.

Identification of the Promoter Region of the bphA Gene

The gene that codes for the enzyme benzoate-para-hydroxylase (BPH) inAspergillus niger was cloned by Van Gorcom et al. (1990) Mol. Gen.Genet. 223, 192-197). The same strain used in developing the presentinvention by the inventors (# ATCC 1015) was obtained from the AmericanType Culture Collection (ATCC), and grown in appropriate culture medium(Pontecorvo (1953) Adv. Genet. 5, 141-238); the DNA from these culturedfungi was isolated as described by Yelton et al. ((1984) Proc. Natl.Acad. Sci. USA 81, 1470-1474). Polymerase Chain Reactions (PCR) werecarried out to confirm that the strain indeed carried the gene encodingBPH.

Primers were designed in such a way that a PCR amplification productfrom genomic DNA could be used as an adequate and specific hybridizationprobe to the bphA gene. The two introns present in the bphA sequencewere excluded, and conserved sequences among cytochrome P450-encodinggenes were avoided when possible. PCR was performed using Pfu DNAPolymerase, the product was electrophoresed on an agarose gel, and DNAfrom the corresponding band was purified using the QIAEX II GelExtraction Kit (Qiagen Inc, Valencia, Calif.), according to themanufacturer's instructions.

This PCR product was cloned into the Bluescript SK vector (StratageneCloning Systems, La Jolla, Calif.) and sequenced at the Iowa StateUniversity Sequencing Facility, to confirm that it is indeed the bphAsequence published by van Gorcom et al. ((1990) Mol. Gen. Genet. 223,192-197). The fragment contained within this vector is designated BPHprobe.

In order to verify whether this or a similar gene is also present inother organisms, a Southern (DNA) blot was performed containing genomicDNA samples of different fungal and plant species. The fungal speciesincluded Aspergillus niger, Aspergillus nidulans A. nidulans was used insubsequent studies because a genome sequencing project is currentlyunderway on this species. The representative plant species included:rice (Oryza sativa), as an example of a grass species; Arabidopsisthaliana, currently a model in plant genetic studies, and with itsentire genome sequenced; and tobacco (Nicotiana tabacum), for which abenzoate-2-hydroxylase activity was reported a few years ago (León etal. (1993) Plant Physiol., 103: 323-328; León et al (1995) Proc. Natl.Acad. Sci. USA, 92: 10413-10417). For the Southern blot, genomic DNAsamples from the species described above were digested with BstEII andXbaI restriction enzymes, separated by electrophoresis on an agarose geland then transferred to Hybond-N⁺ nylon membranes (Amersham Life Sci.Inc, Arlington Heights, Ill.). The BPH probe was radiolabeled(α-³²P-dCTP) by means of random priming, using the Amersham's RediprimeII labeling system; the labeled probe was allowed to hybridize to themembrane for at least 12 hours. Transfer and hybridization procedureswere carried out as recommended by the manufacturer (Amersham).

The time course gene expression induction was examined by analyzingtotal RNA at different times after adding benzoic acid. Northern (RNA)blots were carried out using total RNA extracted from mycelia grown fordifferent periods of time in the presence of benzoic acid. Initially,the fungus was inoculated in CM medium (Pontecorvo (1953) Adv. Genet. 5:141-238) for 24 hours, transferred to benzoate-containing medium (0.1%w/v) for different times, and total RNA was extracted using the RneasyPlant Total RNA kit (Qiagen Inc, Valencia, Calif.) according to themanufacturer's instructions. Total RNA was resolved on aformaldehyde-agarose gel and transferred to a Hybond-N nylon membrane(Amersham Life Sci. Inc, Arlington Heights, Ill.). Radiolabeled BPHprobe was allowed to hybridize to the membrane, as described above.Northern blot methods were also carried out as recommended (Amersham).

Different benzoate concentrations and inducers were used to bettercharacterize the inducibility of the bphA gene. Gene expression wasassayed by Northern blots. Benzoate 4-hydroxylase activity is alsoassessed by enzymatic assays to confirm the inducibility at thetranscriptional level described by Van Gorcom et al. ((1990) Mol. Gen.Genet. 223, 192-197). Inducibility is also verified by reversetranscription of mRNAs produced under induction conditions.

Initial attempts to isolate the promoter region of the bphA gene wereundertaken by utilizing inverse PCR (IPCR) (Ochman et al. (1988); (1993)Meth. Enzym. 218: 309-321) Genetics 120: 621-623). This techniqueconsists of digesting genomic DNA with several enzymes that cut oncewithin the known sequence, and downstream of the region where the probebinds. A Southern (DNA) blot is carried out to identify the enzymes thatproduce a fragment with a reasonable size that might contain thepromoter region. The genomic DNA is then digested with the selectedenzyme, and the resulting fragments are circularized with T4 DNA Ligaseunder very low DNA concentrations. Specific PCR primers are designedsuch that amplification extends toward the region of unknown sequence.The PCR product containing the gene and the promoter region is thencloned into a vector. This approach was initially unsuccessful.

In alternative approaches, the BPH promoter is isolated throughscreening of an A. niger genomic library using the BPH probe, and morefine mapped using restriction endonucleases, or by anchored PCR. Thelatter method was successful.

Promoter deletion studies were also conducted to identifybenzoate-responsive boxes within the BPH promoter region.

Promoter Fusions to GFP

In order to determine the minimal promoter length capable of retainingbenzoic acid inducibility, promoter fusions to the Green FluorescentProtein (GFP) gene were prepared. The full bphA promoter and shorterversions of it were PCR amplified using primers BPHPFwd(5′-GCATTGAAGCTTTACATCGGCCTGACG-3′, SEQ ID NO:19), BPHFR6(5′-AAGCCCAAGCTTGAGTAAGTAAGGAGTTGG-3′, SEQ ID NO:20), BPH331(5′-TTTTCCAAGCTTCCGTAACTCTCGCCTC-3′, SEQ ID NO:21), and BPHPRev(5′-CCCGTTAAGCTTTTTGAGTTGAAGTGCAGG-3′, SEQ ID NO:22). These primersintroduced HindIII restriction sites (underlined) at both ends, whichallowed cloning of the fragments upstream of the GFP gene containedwithin the pEBFP vector (Clontech, Palo Alto, Calif.).

The fragment resulting from the amplification with primers BPHPFwd andBPHPRev corresponded to the full bphA promoter, whereas amplificationwith BPHFR6 and BPHPRev primers resulted in a fragment encompassing basepair −531 to +1, which included FR6 and FR7 mini-fragments.Amplification with primers BPH331 and BPHPRev produced a fragment of theBPH promoter that encompassed base pair −331 to +1, and did not includethe previously identified BREF51. The promoter-GFP fusions were calledpBPH-1847-GFP, pBPH-531-GFP, and pBPH331-GFP, respectively. Theselectable marker Pyr4 gene from Neurospora crassa, driven by its ownpromoter, was used to select putative A. nidulans transformed clones.The selectable marker was PCR-amplified from the pBS-Pyr4 vector usingprimers PyrEcoF (5′-GCAGGAATTCGATCTGCTTCCTCAACC-3′, SEQ ID NO:23), andPyrEcoR (5′-CCGGAATTCGATAAGCTTGATGGGGATC-3′, SEQ ID NO:24), whichintroduced EcoRI restriction sites (underlined) on both ends of theproduct. These sites were used for cloning of the EcoRI-digested productinto the pBPH-GFP deletion constructs described above.

Protoplast Preparation

Aspergillus nidulans GR5 protoplasts were prepared according to Aramayoand Timberlake ((1993) EMBO J. 12: 2039-2048), with some modifications.In summary, conidia were inoculated on CM plates supplemented with 10 mMuracil and incubated for 2 d at 37° C. Approximately 10⁹ fresh conidiawere aseptically collected from plates and inoculated into 250 mL ofliquid CM medium+10 mM uracil. After overnight incubation at 30° C., 150rpm, mycelia were harvested through a sterile Büchner funnel lined withtwo layers of Miracloth, and washed twice with Mycelium Wash Solution(0.6 M MgSO₄). Washed mycelia were then suspended in 40 mL of freshlyprepared Osmotic Media (1.2 M MgSO₄, 10 mM sodium phosphate buffer, pH5.8), containing 40 U β-glucuronidase (Sigma) and 0.4 g Lysing Enzymesfrom Trichoderma harzianum (Sigma), and incubated for approximately 3 hat 28° C., 120 rpm. The resulting protoplasts were filtered through aBüchner funnel lined with three sheets of sterile Miracloth into a 50 mLconical centrifuge tube, and one volume of ice-cold STC50 solution (1.2M sorbitol, 10 mM CaCl₂, 50 mM Tris-HCl, pH 7.5) was added. Protoplastswere collected by centrifugation at 2,000×g for 10 min, at 4° C. Asecond wash was performed by suspending the pelleted protoplasts in 1 mLof STC50 and centrifuging again as described above. Concentration ofprotoplasts was determined using a hemacytometer, and adjusted to 10⁸protoplasts per mL.

Protoplast Transformation

The procedure described by Rolf Prade, Oklahoma State University, on theWeb Site (microbiology.okstate.edu/faculty/prade/) was used forintroduction of plasmid DNA into A. nidulans GR5 protoplasts. Briefly,approximately 10 μg of plasmid DNA dissolved in 10 mM Tris-HCl (pH 8.0)were added to 100 μL protoplasts (approx. 10⁷ protoplasts), and themixture was incubated on ice for 10 min. Next, 250 μL of freshlyprepared 60% PEG 3350 (Sigma) in STC50 were added, and incubation wascarried out at 37° C. for an additional 20 min. After addition of 2 mLSTC50 containing 1% glucose and gently mixing, protoplasts were platedonto molten minimal medium, containing 1.2 M sorbitol, and incubated at37° C. until uracil auxotrophic colonies became visible.

Identification of Benzoate Inducible Transcription Factors and GenesEncoding Them

Promoter activity is usually modulated by protein factors that bind tothe DNA and either activate or repress transcription. Thus, asnecessary, proteins (transcription factors) that bind to the promoterand regulate transcription of the bphA gene are also identified. Theseexperiments utilize gel-shift assays as described by Singh et al.((1986) Nature 319: 154-158). In order to investigate whether anyproteins bind to the bphA promoter, EMSAs were carried out in thepresence of total protein extracts from A. niger mycelia. This assay isbased on the fact that free double-stranded DNA fragments will runfaster on a native polyacrylamide gel than those that have proteinsassociated to them. The association results in a shift in the mobilityof the DNA fragment on the gel.

Genes that code for proteins involved in the regulation of the BPHpromoter are cloned as well. Benzoate-inducibility of the transcriptionfactor is tested.

Total Protein Extraction.

Total protein extracts were prepared from both benzoate-induced anduninduced A. niger mycelia according to the procedure described byPeters and Caddick (1994) Nucl. Acids. Res. 22, 5164-517), with a fewmodifications. Briefly, mycelia were ground in liquid N₂ and theresulting powder was suspended in Extraction Buffer (20 mM HEPES-KOH [pH7.9], 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 2 mM DTT, proteaseinhibitor cock-tail (Sigma cat# P2714) (5 ml/g of tissue). Saturatedammonium sulfate solution (pH 7.0) was added to the lysate to a finalconcentration of 0.4 M. After stirring for 15 min, the solution wasallowed to stand for another 15 min. Cell debris and chromosomal DNAwere removed by centrifugation at 100,000×g, 4° C., for 30 min, andsolid ammonium sulfate was added to the supernatant over a period of 90min, to 70% saturation, while stirring gently. Again, the lysate wasallowed to stand for a further 30 min, and precipitated proteins werepelleted by centrifugation at 10,000×g, 4° C., for 30 min. The pelletwas resuspended in Binding Buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl,0.5 mM DTT, poly [dI-dC]), and the solution was dialyzed overnightagainst the sane Binding Buffer. Protein concentration was determined bythe Bradford method (Bio-Rad), and bovine serum albumin (BSA) was usedas a standard.

Electrophoretic Mobility Shift Assays (EMSA).

Promoter activity is usually modulated by protein factors that bind tothe DNA and either activate or repress transcription. In order toinvestigate whether any proteins bind to the bphA promoter, EMSAs werecarried out in the presence of total protein extracts from A. nigermycelia. This assay is based on the fact that free double-stranded DNAfragments will run faster on a native polyacrylamide gel than those thathave proteins associated to them. The association results in a shift inthe mobility of the DNA fragment on the gel.

The BandShift Kit (Pharmacia Biotech, Piscataway, N.J.) was used forthis purpose. DNA mini-fragments from the bphA gene promoter region wereprepared by means of PCR, separated on a 0.8% agarose gel, and purifiedusing the QIAEX II Gel Extraction Kit (Qiagen, Valencia, Calif.).Approximately 100 ng of each mini-fragment were [g³²P]dATP-labeled usingT4 polynucleotide kinase enzyme, purified through a Sephadex G-25column, and mixed with 1 mg of total protein extract, along with 5%glycerol, 0.05% NP-40, 1 mg Poly(dI-dC)-Poly(dI-dC), 10 mM Tris-HCl (pH7.5), 50 mM NaCl, and 0.5 mM DTT. This mixture was incubated for 1 to 3h at room temperature, and then electrophoresed on 5% polyacrylamidegels under non-denaturing conditions. DNA mobility shifts werevisualized after exposing the gels to an X-ray film.

Purification of putative transcription factors. Transcription factorsare purified by one of two approaches, affinity chromatography and/or amolecular approach.

1. Affinity Chromatography

In one approach, putative transcription factors are purified using BRE51fragment as “bait” for affinity chromatography:

Poly(A) tail method. Sense and anti-sense strands, corresponding to theBRE51 fragment sequence, were synthesized at IDT DNA Technologies with a3′ poly(A) overhang. Both strands were annealed by mixing 1 mg of eachtogether in 100 ml of 50 mM NaCl and heating the mixture to 70° C. for 5min, then slowly cooling it down to room temperature for at least 30min. After electrophoresing the annealed fragment on a 2% Low MeltingPoint Agarose gel, the desired double-stranded fragment was purifiedfrom the gel using the QIAEXII Gel Purification Kit (Qiagen, Valencia,Calif.) according to the manufacturer instructions. The double strandedBRE51 fragment, containing the poly(A) overhang was then called BREaff.

Approximately 1 mg of BREaff was annealed at room temperature to aBiotinylated-Oligo(dT) probe (PolyATract® mRNA Isolation System,Promega, Madison, Wis.), and the mixture was added to a tube containingStreptavidin coupled to Paramagnetic Particles (SA-PMPs) in 0.5×SSC (1×is 150 mM NaCl, 15 mM Na₃citrate). After washing the particles once with0.1×SSC, and twice with 1× Binding Buffer (10 mM Tris-HCl pH 7.5, 50 mMNaCl, 0.5 mM DTT, poly [dI-dC]), 10 mg of benzoate-induced total proteinextracts were added and the reaction was incubated at room temperaturefor 2 h. After two washes with 1× Binding Buffer (without poly[dI-dC]),BREaff bound to proteins was eluted from SA-PMPs with 100 ml of water.Eluted proteins were separated on 12% SDS-PAGE and visualized byCoomassie blue staining.

Biotin labeling method. An alternative approach to the affinitychromatography described above was devised by labeling BRE with Biotin.Briefly, BRE51 was PCR-amplified and an EcoRI restriction site was addedat the 5′ side. After EcoRI digestion of the product, the overhang wasfilled in by E. coli DNA Polymerase Klenow fragment (New EnglandBioLabs) at 25° C., for 15 min, in the presence of 33 mM each of dCTP,dGTP, dTTP, and Biotin-7-dATP (Gibco BRL). Biotinylated BRE51 waspurified with QIAEX II Gel Extraction kit (Qiagen, Valencia, Calif.),and added to the streptavidin-coupled paramagnetic particles, asdescribed above. Washes and incubation with total proteins were carriedout exactly as described above, but bound proteins were eluted fromSA-PMPs with binding buffer containing 1 M NaCl.

2. Molecular Approach for the Isolation of the Transcription FactorGene: Activation Domain-cDNA Library Construction

Total RNA was extracted from A. niger cells grown in the presence ofbenzoic acid for 5 h by using the RNAeasy kit (Qiagen), following theinstructions of the manufacturer. Ten μg of total RNA were used toisolate poly A⁺ RNA, using the PolyATtract kit (Promega, Madison, Wis.),also according to the instructions provided. Complementary DNAs (cDNAs)were synthesized using the Matchmaker Library Construction and ScreeningKit (Clontech, Palo Alto, Calif.), which employs the SMART™ technology.First strand cDNAs were synthesized at 42° C. for 10 mm, fromapproximately 1 μg of poly A⁺ RNA, using 1 μM Oligo d(T) primer (CDSIII,5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)₃₀VN-3′) (SEQ ID NO:38) and MMLVReverse Transcriptase. One μM SmartIII™ oligonucleotide(5′-AAGCAGTGGTATCAA CGCAGAGTGGCCATTATGGCCGGG-3′, SEQ ID NO:25) wasadded, and was the reaction incubated at 42° C. for an additional 1 h.After removing DNA-RNA double strands with RNAse H, double strandedcDNAs were amplified by Long Distance-PCR (LD-PCR). Thermocycler cyclesconsisted of incubation at 95° C. for 30 sec, followed by 20 cycles,each consisting of denaturation at 95° C. for 30 sec and combinedannealing and extension at 68° C. for 6 mm. Extension time increased by5 sec each cycle. PCR-amplified ds cDNAs were purified through a ChromaSpin+TE400 size exclusion column.

The library was constructed by in vivo recombination of cDNAs in yeast.Competent yeast cells strain AH109 were prepared and transformed withthe purified ds cDNAs and SmaI-linearized pGADT7-Rec vector. In vivorecombination yields a complete GAL4 activation domain vector.Transformants were selected on SD/-Leu media.

Yeast One-Hybrid Screen

A reporter construct was prepared by introducing three tandem copies ofthe previously identified BREF51 immediately upstream of a minimalpromoter in vector pHISi-1 (Clontech, Palo Alto, Calif.). Tandem copiesof BREF51 were first assembled in a pBluescript SK (−) vector. BREF51was PCR-amplified using primers BREBP (5′-TATGGATCCCTGCAG,ACTAGTCACAAGTTAC-3′, SEQ ID NO:26) and BREBN(5′-TATGGATCCATGCATGATCTTCATGACTAATGATG-3′, SEQ ID NO:27), whichintroduced BamHI and PstI restriction sites at the 5′ end (underlined onBREBP), as well as BamHI and NsiI sites at the 3′ end (underlined onBREBN). After cloning the amplified BREF51 into pBluescript (namedpBSF51), one aliquot of this plasmid was digested with NsiI and AflIIIenzymes, and another aliquot with PstI and AflIII enzymes, the twocomplementary fragments were ligated to each other, reconstructing acomplete vector containing 2 copies of BREF51, which was called pBSF102.This procedure was iterated, this time using pBSF102 as one of thealiquots. The final vector containing 3 copies of BREF51 was then calledpBSF153. The 3 tandem copies of BREF51 were then sub-cloned to pHisi-1vector using EcoRI and XbaI restriction sites. The resulting chimericpromoter in pHisi-1 controls expression of the HIS3 gene from yeast,which encodes the enzyme imidazole glycerol-phosphate dehydratase. Thisvector was then called pHISi-1.153, and was integrated into thechromosome of yeast strain YM4271 by first linearizing and thentransforming into competent cells following the small-scale LiActransformation procedure (Ito et al. (1983) J. Bacteriol. 153: 163-168)to produce the reporter strain YM4271HIS. Leaky expression from thechimeric promoter allowed selection of clones containing the integratedvector. To control leakiness of the promoter during the one-hybridscreen, 30 mM 3-AT were added to the culture media. This concentrationhas been shown in a concentration curve experiment to completely abolishgrowth of the reporter strain in the absence of histidine (data notshown).

Plasmid DNA isolated from approximately 1×10⁶ AD-cDNA library colonieswere transformed into YM4271HIS following the procedures described byGietz et al. ((1992) Nucl. Acids Res. 20: 1425). Briefly, anovernight-grown culture of YM4271HIS (OD₆₀₀=0.5) was centrifuged for 5min at 1000×g, and resuspended in TE buffer (10 mM Tris.HCl, pH 8.0, 1mM EDTA). After another centrifugation step as described above,competent cells were resuspended in freshly prepared 1× TE/LiAc. In asterile tube, 20 μg of AD-cDNA library plasmid were mixed with 2 mg ofherring testes carrier DNA, before 1 mL of competent cells were addedand mixed. Six mL of sterile PEG/LiAc were added to the transformationmixture, mixed by vortexing, and incubated at 30° C. for 30 min withshaking at 200 rpm. DMSO was added (700 μL), mixed by inversion, andcells were heat-shocked for 15 min at 42° C. After chilling on ice for 2min, the mixture was centrifuged for 5 min at 1000×g, at ambienttemperature, and resuspended in TE buffer to a total of 7.5 mL. Thetransformation mixture was plated on 150×15 mm Petri dishes (500μL/plate) containing SD/-Leu/-His media supplemented with 30 mM 3-AT, toselect for clones displaying DNA-protein interaction. Plates wereincubated at 30° C. until colonies were visible.

Plasmid Purification from Positive Clones

Plasmid DNA was purified from positive clones selected on the one-hybridscreen by using Clontech's Yeast Plasmid Isolation Kit. Liquid culturesgrown overnight at 30° C. were centrifuged and the supernatant wasresuspended in 50 μL of 67 mM KPO₄ buffer (pH 7.5). Fifty units ofLyticase were added and the mixture incubated at 37° C. for 60 min.After 10 μL of 20% SDS were added and the tubes vortexed vigorously for1 min, plasmid DNA was purified using Chroma Spin-1000 DEPC-H₂O columns(BD Biosciences, Palo Alto, Calif.).

cDNA Insert Size Determination

PCR amplifications using primers specific for the pGADT7-Rec vector(GADF: 5′-CTATTCGATGATGAAGATACCCCACC-3″, SEQ ID NO:28, and GADR:5′-GTGAACTTGCGGGGTTTTTCAG-3′, SEQ ID NO:29) were carried out with theobjective of determining the size of the cDNA insert. PCR conditionswere as follows: denaturation at 94° C. for 5 min, followed by 25cycles, each consisting of denaturation at 94° C. for 1 min, annealingat 56° C. for 45 sec, and extension at 72° C. for 1 min. One final cycleof extension at 72° C. for 10 min was also used. Amplified products wereseparated on a 0.8% agarose gel containing EtBr, and the bandsvisualized under UV light.

E. coli Transformation of Yeast Plasmids

To obtain enough plasmid DNA for sequencing, 5-10 μL of yeast plasmidDNA isolated from one-hybrid positive clones were transformed into E.coli XL1-Blue competent cells by electroporation in 1 mm-gap cuvettes,and using standard procedures (Sambrook et al. (1989) Molecular Cloning:A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor (N.Y.) LaboratoryPress, Vol. 1). Plasmid DNA was isolated from bacterial cells using theQuantum Prep Plasmid Mini Prep kit (Bio-Rad, Hercules, Calif.),according to the instructions provided by the manufacturer.

cDNA Sequencing

cDNA inserts that yielded at least one product when amplified by PCRwere submitted for sequencing at the Low Throughput Sequencing Center,at the Purdue University Genomics facility. DNA sequence was obtainedfrom the 5′ end of the cDNAs using the GADF primer. When necessary,sequence from the 3′ end was also obtained using primer GADR. Thenucleic acid sequence for GAD1 (SEQ ID NO: 16) is shown in FIG. 16, thenucleic acid sequence for GAD11 (SEQ ID NO:18) is shown in FIG. 17, thenucleic acid sequence for a GAD1 homolog (SEQ ID NO:35) is shown in FIG.18, and the nucleic acid sequence for a GAD11 homolog (SEQ ID NO:37) isshown in FIG. 19.

Total Protein Extraction from Yeast

Positive clones identified on the one-hybrid screen were grown in 100 mLof SD/-Leu media, and total proteins were extracted following a modifiedprocedure from that described by Arndt et al. ((1987) Science 237:874-880). All extraction steps were performed at 4° C. Briefly, cellswere pelleted by centrifugation at 1000×g for 5 min, and resuspended in400 μL of protein extraction buffer (0.1 M Tris.HCl pH 7.5, 0.2 M NaCl,0.01 M β-mercaptoethanol, 20% glycerol, 5 mM EDTA, 1 mM PMSF). Glassbeads (425-600 microns in diameter) were added and tubes were vortexedvigorously for 10 min in a Geno/Grinder 2000 (SPEX CertiPrep, Metuchen,N.J.). The glass beads were allowed to settle, and the supernatant wastransferred to a new tube. New glass beads and 200 μL of the sameprotein extraction buffer were added, and the tubes were vortexed againas described. Protein concentration of the resulting supernatant wasdetermined by the Bradford method (Bio-Rad, Hercules, Calif.), and BSAwas used as a standard.

Reverse Transcription PCR

Total RNA was extracted from uninduced and benzoic acid-induced A. nigermycelia as described earlier, and approximately 1 μg was used tosynthesize first strand cDNAs with the SuperScript First-strandSynthesis System for RT-PCR (Invitrogen, Carlsbad, Calif.). The reactionwas carried out in the presence of 0.5 μg of an oligo(dT) primer, 0.5 mMdNTP mix, 5 mM MgCl₂, 10 mM DTT, 1× RT buffer (20 mM Tris.HCl pH 8.4, 50mM KCl), RNAseOUT™ RNAse inhibitor, and 50 U of SuperScript™ II ReverseTranscriptase. Tubes were incubated at 42° C. for 50 min, after whichthe reaction was terminated at 70° C. for 15 min. RNAse H was added, andtubes were incubated for an additional 20 min at 37° C. Double-strandedcDNAs were amplified by PCR using Red Taq DNA Polymerase (Sigma-Aldrich,St. Louis, Mo.), dNTP's, and gene specific primers: GAD1F(5′-CACATACACAATGGTCTCCTTCAAG-3′, SEQ ID NO:30), GAD1R(5′-CTGCACACATGTAATACGCATACC-3′, SEQ ID NO:31), GAD11F(5′-GGTTTAGCCTTCACTCTCAAGGATC-3′, SEQ ID NO:32), and GAD11R(5′-GTCGAAAGGTGCGATTCGATATAGG-3′, SEQ ID NO:33). PCR amplificationconsisted of a denaturation step at 94° C. for 5 min, followed by 30cycles, each of denaturation at 94° C. for 45 sec, annealing at 56° C.for 30 sec, and extension at 72° C. for 1 min. A final extension step at72° C. for 10 min was also included. Amplified cDNAs were separated on a0.8% agarose gel containing EtBr, and bands were visualized under UVlight.

Construction of a Benzoate Inducible Hybrid Promoter

A chemically-induced promoter (hybrid promoter) is constructed byplacing benzoate-response elements within plant regulatory regions, tomake it functional in plants.

A chemically inducible promoter is constructed by placing single ormultiple copies of BREF51, or functionally effective fragments ormodifications thereof, upstream of a minimal plant gene promotercontaining a TATA box transcription start signal, to make the benzoateresponse element functional in cells; the product is referred to as abenzoate inducible hybrid promoter. In some embodiments, additionalplant gene promoter elements may be placed around the BREF51, orfunctionally effective fragments or modifications thereof, to achieve ahost cell-specific temporal or spatial expression in response to thechemical application.

This hybrid promoter is fused to a reporter gene, such as GUS (Jeffersonet al., (1987) EMBO J. 6: 3901-3907) or GFP (Haseloff et al. (1997)Proc. Natl. Acad. Sci. USA 94: 2122-2127), and transformed intoArabidopsis thaliana by Agrobacterium-mediated transformation asdescribed by Ye et al. ((1999) Plant J. 19: 249-257). Another plasmidcontaining the gene that codes for the transcription factor isintroduced into these plants as well, if necessary. The transgenicplants obtained are assayed for the inducibility of expression of thereporter gene. Northern blots are carried out to verify both thebackground and the induced levels of expression of the reporter gene inthe plant, under different concentrations of the inducer. The effects,if any, of the inducible system on the overall performance of the plantsare also evaluated. Evaluated characteristics include but are notlimited to pathogen or insect resistance, whole plant and tissueorganization, flowering time, or any other signs of toxicity.

Example 2 Characterization of Benzoate Response Elements, Promoters, andTranscription Factors

This example describes results of the development of the presentinvention, and includes characterization of the bphA gene expression,identification and isolation of bphA gene promoter and response element,and identification of transcription factors

Characterization of bphA Gene Expression

In order to verify whether this or a similar gene is also present inother organisms, a Southern (DNA) blot was performed containing genomicDNA samples of different fungal and plant species. A genomic DNA blotfrom samples of Aspergillus niger, A. nidulans, tobacco, rice andArabidopsis was probed with α-³²P-dCTP-labeled BPH probe; the resultsare shown in FIG. 2. As expected, a band of 1100 base pairs was labeledby the BPH probe after restriction digestion with the BstEII enzyme.Aspergillus nidulans also seems to contain a similar gene in its genome,as shown by the >12 kbp band produced. The coding region of the bphAgene does not contain any restriction sites for the XbaI enzyme andtherefore is a good way of determining the number of copies of this genein the genome. Cleavage of genomic DNA with a restriction enzymes can beexpected to fragment different genes at different places to give a rangeof sizes. Use of several enzymes confirms the number of independentgenes. However, some enzymes will cut within the coding region to yieldtwo fragments instead of one. In instances where a single band isobserved after cutting with several enzymes, then one can be reasonablyconfident there is only one copy of the gene (in other words, nohomologue). The results indicate that both fungi have only one copy ofthe gene. None of the plant species used in this experiment appeared tocontain a gene similar in nucleic acid sequence to the bphA gene.

To verify the inducibility of the bphA gene by benzoic acid, A. nigerwas inoculated in complete medium and grown for 24 hours before beingtransferred to fresh medium containing 0.1% benzoic acid. Mycelia wereharvested after different periods of induction, and total RNA wasextracted. A preliminary RNA blot showed that the bphA gene is inducedat the transcriptional level within ten minutes by benzoic acid (FIG.3).

Concentration curves indicate that benzoic acid concentrations as low as0.8 mM induce transcription of the bphA gene. Other compounds which caninduce the promoter include sodium benzoate and methyl benzoate;however, benzyl alcohol and hydrocinnamic acid do not induce thepromoter (FIG. 4).

Identification and Isolation of bphA Gene Promoter and Response Element

Using Anchored Polymerase Chain Reactions (PCR) (Siebert et al. (1995)Nucl. Acids Res. 23: 1087-1088), about 1.8 kb of the promoter region ofthe Aspergillus niger bphA gene was isolated, and then cloned intopBluescript SK⁻. The sequence of the 1.8 kb of the promoter region ofthe Aspergillus niger bphA gene (SEQ ID NO:1) is shown in FIG. 1.

In order to investigate whether protein factors are involved incontrolling the initiation of transcription from the bphA gene promoter,seven mini-promoter fragments were created by PCR (FIG. 5), ranging insize from 247 bp to 324 bp. These fragments were thenradioactive-labeled with ³²P-dATP, and used in ElectrophoreticMobility-Shift Assays (EMSA). After confirming that one of thesefragments (FR6, 323 bp) was bound by protein factors (FIG. 6), partiallyoverlapping sub-fragments encompassing the FR6 fragment were prepared,and again used in EMSA. These assays showed that a 51-bp fragmentlocated 350 bp upstream of the transcription start point is consistentlybound by a protein factor that is present in total protein extracts frombenzoate-induced A. niger mycelia (FIG. 7). The most significant featureof this BRE sequence (shown in FIG. 8) is the presence of a pair of 6-bpdirect repeats of the potential BRE (underlined in FIG. 8).

bphA gene promoter fusions to the Green Fluorescent Protein (GFP) genewere constructed and transformed into Aspergillus nidulans strain GR5(ATCC # 200171). Both the full promoter (1.8 kbp) and a short versioncontaining fragment 6 and the TATA box (FR6, 0.4 kbp) were placed infront of the GFP coding region (pEBFP, Clontech, Palo Alto, Calif.).Transformants were screened by PCR for the presence of the GFP gene, andgrown either in the presence or in the absence of 8 mM benzoic acid.After 5 hours of induction, both promoters were able to induce GFPexpression only in the presence of the inducer benzoate (FIG. 8).

Identification of Transcription Factors

Initial efforts to isolate trans-acting factor(s) involved in theregulation of the bphA gene promoter utilized affinity chromatographyexperiments using either the BRE51 or benzoic acid as affinity ligands.In a first approach, BRE was biotinylated, purified, and allowed to bindto streptavidin-linked paramagnetic particles (PMPs; PolyATtract mRNAIsolation Kit, Promega, Madison, Wis.). Total protein extract frombenzoate-induced A. niger was added to the solution, incubated for 60min, and the PMPs were washed several times. Bound proteins were theneluted from the PMPs under high salt conditions. The fractions wereelectrophoresed on SDS-PAGE, and a protein of approximately 45 kDa couldbe observed (FIG. 10); however, this factor failed to cause a shift onthe mobility of BRE51 when used on EMSA. In a second approach, benzoicacid, immobilized on 4% cross-linked beaded agarose (Sigma, St. Louis,Mo.) was used as the affinity ligand. Again, total protein extract frombenzoate-induced A. niger was added, the column was washed, and boundproteins eluted under high salt conditions. The eluted fraction alsofailed to cause mobility shifts on labeled BRE51 when used on EMSA.

Calculation of the stoichiometry of the amount of binding of a proteinelement that saturates the amount of BREF51 in the EMSA indicates that aminimum of 6 fmoles are present per mg of protein extract. If the factoris about 50 kDa, then this amount represents 300 pg of factor. Thus, amanageable scale-up of about 100-fold is necessary to bind 30 ng offactor needed to detect tryptic fragments by MALDI-TOF MS.

The hypothesis of a surface receptor was tested by using thep-NH₂-benzoate coupled to agarose (as described above) as anon-cleavable external probe. Fungal protoplasts containing the BREfused to GFP were then prepared, and activation of the BRE (productionof the GFP marker) by addition of the agarose-immobilized benzoatederivative to the fungal protoplasts was scored. Whereas the benzoate orp-aminobenzoate induced GFP fluorescence in the BRE-GFP transformedline, no fluorescence was observed when p-nitrobenzoate was linked tothe agarose beads. This initial set of results indicates that the TF isa soluble factor. In order to investigate whether the disparity of sizebetween the larger agarose beads and fungal protoplasts may have causedsteric problems in binding of the inducer, smaller ligands stillincapable of uptake are constructed and utilized to confirm anon-membrane surface location.

The yeast one-hybrid system screen yielded 17 colonies on SD/-Leu/-Hismedia. DNA sequences obtained from the plasmids isolated from thebacterial cells were translated and compared by BLASTp search (Table 1).Total proteins were isolated from 9 yeast clones which cDNAs encodedproteins that did not show significant similarities to any gene of knownfunction in the databases. Protein extracts from each of these yeastclones were used in EMSA to eliminate those cDNAs that encode proteinsthat do not interact with BREF51. Yeast clones GAD1 (FIG. 16) and GAD11(FIG. 17) both showed a gel mobility shift of the BREF51 fragment inthese assays (FIG. 13). The shift in mobility displayed by GAD1 wassimilar to that observed with A. niger total protein extracts. Theprotein encoded by GAD1 is highly similar to a predicted protein presentin the A. nidulans genome (E value 2e-83, see FIG. 18), and similar to ahypothetical protein (B24P11.210) in the N. crassa genome (E value4e-27) (Table 2). GAD1 protein is rich in serine residues (16%), and ispredicted by PSORTII to be localized in the nucleus (70.6% probability).It also shows high similarity to a hypothetical serine-rich protein(C13G6.10c) from S. pombe. Serine-rich motifs can be phosphorylationtargets by protein kinase C in proteins. Also, serine-rich regions havebeen shown to be part of the transactivation domains of sometranscription factors in both mammalian and viral cells. While notnecessary to understand or practice the present invention, if GAD1 isinvolved in the regulation of BPHprom, GAD1 protein might be alwaysbound to a benzoic acid response element in the bphA promoter. In theabsence of benzoic acid GAD1 would be inactive, unable to promotetranscription. However, addition of benzoic acid could causephosphorylation of one of the serine residues in GAD1, leading to itsactivation. This mode of regulation is analogous to that of thetranscription factor CREB, which is involved in responses to cAMP inhuman cells (Latchman 1997).

GAD11 protein (SEQ ID NO:17; FIG. 17) is weakly similar to a Drosophilamelanogaster homeotic gene regulator, and also to a putative nuclearprotein family member from the nematode C. elegans. It is also predictedby PSORTII to be localized in the nucleus (94.1% probability). GAD11 wasalso predicted to have a coiled-coil region consisting of 37 amino acidresidues (Lupa's algorithm). The α-helical coiled coil is a structuralmotif found in many proteins. It consists of two long α-helices with arepeating pattern of hydrophobic side chains that interlock to produce asupercoiled structure. The most important function of coiled-coilregions in myosins and kinesins is for dimerization. Because the coiledcoil may be rigid and extended in solution it can act as a spacer orconnector between protein domains. This may explain the larger shift inBREF51 mobility in the presence of GAD11 protein extract, since GAD11proteins may be forming homodimers connected by the coiled-coil region.GAD11 has a homolog in A. nidulans (E value 6e-29, See FIG. 19), but nohomologs in N. crassa were found.

Reverse Transcription PCR (RT-PCR) experiments revealed that while GAD1is expressed constitutively, GAD11 is slightly downregulated in thepresence of 8 mM benzoic acid for 5 h (FIG. 14). While not necessary tounderstand or practice the present invention, if in fact either GAD1 orGAD11 are involved in regulation of the bphA promoter, constitutivetranscription of GAD1 does not rule out the possibility that theactivity of the GAD1 protein is modulated by benzoic acid. On the otherhand, downregulation of the GAD11 gene in the presence of benzoic acidindicates that the protein may function as a transcriptional repressorof the bphA promoter. GAD1 is constitutively expressed, while GAD11seems to be downregulated in the presence of benzoic acid in the media.There is a possibility that protein-protein interactions play a role inthe regulation of this gene in A. niger.

Example 3 Chemical Induction of Flowering in a Model System

The ability of a chemically controllable promoter to induce floweringwas tested in Arabidopsis containing a disabled FT gene. The ft-3 mutantArabidopsis, which is caused by missense mutation deleting the entireC-terminal region of the protein and displays a significant delay in theflowering response, was obtained from the Arabidopsis Stock Center (OhioState Univ).

A chemically inducible promoter available to test induction of floweringis the glucocorticoid receptor-promoter element system that is inducedby the glucocorticoid, dexamethasone (Aoyama and Chua (1997) Plant J.11: 605-612; Kunkel et al. (1999) Nature Biotech. 17: 916-919). Thissystem has recently been engineered to avoid some leakiness problemsoriginally experienced, and the plasmid constructs for the two necessarycomponents have been obtained. The general strategy of theglucocorticoid receptor promoter was described by Aoyama and Chua((1997) Plant J. 11, 605-612). Dr. Jen Sheen (Massachusetts Generalhospital) restructured the glucocorticoid promoter to be LexA-basedinstead of Gal4, and this provided a finer control. This promoter can beassembled from available sequences by one skilled in the art.

Wild-type FT cDNA was obtained from Dr. Detlef Weigel (formerly of theSalk Institute, La Jolla, Calif., and now Max Planck Institute forDevelopmental Biology, Tübingen, Germany); the wild-type FT cDNA wasprepared from transcripts for flowering Arabidopsis plants. This andother Arabidopsis cDNAs are available from Dr. Weigel or from a publiclyaccessible Riken BioResource Center, Japan (See Riken Web site). As theArabidopsis genome is completely sequenced and publicly available, oneskilled in the art can design primers and obtain a full-length FT geneby RT-PCR. Wild-type FT cDNA was placed under the control of theLexA-based glucocorticoid inducible promoter (called Lex-FT), and thisconstruct was subcloned into the pCAMBIA 1305.1 binary vector (CAMBIA,Canberra, Australia). In a separate construct, the hybridtransactivating factor LSVG, under a constitutively expressed promoter,was cloned into pCAMBIA 1302 (CAMBIA, Canberra, Australia). Both vectorswere then transformed by vacuum infiltration (Clough and Bent (1998)Plant J. 16: 735-743) into the Arabidopsis ft plants. T1 seeds wereharvested, and plated out on selective media, containing the antibiotichygromycin, in order to recover putative transformants.

The selected transformants have been assayed for GUS or GFP expression(depending on the pCAMBIA vector used), and the presence of theinducible promoter system and the FT gene have been confirmed by PCR. Aproblem was encountered with the plants containing the pCAMBIA 1302-LSVGconstitutive-expressant construct, probably due to a slow accumulationof toxic levels the hybrid transacting factor LSVG during the prolongedgrowth before flowering. The transgenic plants died shortly afterflowering, yielding few or no seeds from the plants. To overcome thisunexpected difficulty with this test system, the LSVG hybridtransactivator was subcloned into a different pCAMBIA vector, with akanamycin selectable marker instead of hygromycin. Confirmed Lex-FThomozygous plants are transformed with the new construct. This approachprovides not only a faster way of incorporating both components into thesame plant, but also overcomes the toxicity problem by spraying the testplants after only one-third the growth period.

As described above, the Lex-FT construct has been successfullyintegrated into non-leaky ft homozygous mutants; these plants are thentransformed with the altered construct containing a constitutivelyexpressed LSVG in a binary vector with kanamycin resistance.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry, and molecular biology or related fields are intended to bewithin the scope of the following claims.

1. A recombinant plant expression vector comprising: a) a benzoateinducible promoter comprising a benzoate response element comprising afragment of SEQ ID NO:1 comprising SEQ ID NO:4 operably linked to apromoter functional in a plant, and b) a nucleic acid sequence ofinterest, wherein said nucleic acid sequence of interest is operablylinked to said benzoate inducible promoter.
 2. The recombinant plantexpression vector of claim 1, wherein said promoter comprises a plantminimal gene promoter.
 3. The recombinant plant expression vector ofclaim 1, wherein said promoter is 35S.
 4. The recombinant plantexpression vector of claim 1, wherein the fragment of SEQ ID NO:1comprising SEQ ID NO:4 is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, or SEQ ID NO:6.
 5. A transgenic plant cell comprising: a) abenzoate inducible promoter comprising a benzoate response elementcomprising a fragment of SEQ ID NO:1 comprising SEQ ID NO:4 operablylinked to a promoter functional in a plant, and b) a nucleic acidsequence of interest, wherein said nucleic acid sequence of interest isoperably linked to said benzoate inducible promoter.
 6. The transgenicplant cell of claim 5, wherein said promoter comprises a plant minimalgene promoter.
 7. The transgenic plant cell of claim 5, wherein saidpromoter is 35S.
 8. The transgenic plant cell of claim 5, wherein thefragment of SEQ ID NO:1 comprising SEQ ID NO:4 is SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6.
 9. A transgenic plantcell comprising: a) a benzoate inducible promoter comprising a benzoateresponse element comprising a fragment of SEQ ID NO:1 comprising SEQ IDNO:4 operably linked to a promoter functional in a plant, and b) anucleic acid sequence of interest, wherein said nucleic acid sequence ofinterest is operably linked to said benzoate inducible promoter.
 10. Thetransgenic plant cell of claim 9, wherein said promoter comprises aplant minimal gene promoter.
 11. The transgenic plant cell of claim 9,wherein said promoter is 35S.
 12. The transgenic plant cell of claim 9,wherein the fragment of SEQ ID NO:1 comprising SEQ ID NO:4 is SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:6.
 13. Amethod of controlling expression of a nucleic acid sequence of interestin a plant, the method comprising: a) transforming said plant with anexpression cassette comprising i) a benzoate inducible promotercomprising a benzoate response element comprising a fragment of SEQ IDNO: 1 comprising SEQ ID NO:4 operably linked to a promoter functional ina plant, and ii) a nucleic acid sequence of interest, wherein saidnucleic acid sequence of interest is operably linked to said benzoateinducible promoter; and b) contacting said transformed plant withbenzoate, whereby said benzoate inducible promoter in the presence ofsaid benzoate induces the expression of said nucleic acid sequence ofinterest.
 14. The method of claim 13, wherein said promoter comprises aplant minimal gene promoter.
 15. The method of claim 13, wherein saidpromoter is 35S.
 16. The method of claim 13, wherein the fragment of SEQID NO: 1 comprising SEQ ID NO:4 is SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, or SEQ ID NO:6.